Phi-4 polypeptides and methods for their use

ABSTRACT

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes. The insecticidal proteins find use in controlling, inhibiting growth or killing lepidopteran, coleopteran, dipteran, fungal, hemipteran, and nematode pest populations and for producing compositions with insecticidal activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non Provisional applicationSer. No. 13/839,702 filed Mar. 15, 2013 the contents of which are hereinincorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“5192USCNT_Sequence_Listing” created on Jun. 8, 2016, and having a sizeof 3632 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190309321A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Lengthy Tables

The patent application contains a lengthy table section as an ASCIIformatted document, which is part of the specification and is hereinincorporated by reference in its entirety. A copy of the tables areavailable in electronic form from the USPTO web site(http://seqdata.uspto.gov/). An electronic copy of the table will alsobe available from the USPTO upon request and payment of the fee setforth in 37 CFR 1.19(b)(3)

FIELD OF THE INVENTION

This disclosure relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These pesticidal proteinsand the nucleic acid sequences encoding them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

Biological control of insect pests of agricultural significance using amicrobial agent, such as fungi, bacteria, or another species of insectaffords an environmentally friendly and commercially attractivealternative to synthetic chemical pesticides. Generally speaking, theuse of biopesticides presents a lower risk of pollution andenvironmental hazards, and biopesticides provide greater targetspecificity than is characteristic of traditional broad-spectrumchemical insecticides. In addition, biopesticides often cost less toproduce and thus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a range of insect pests includingLepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillusthuringiensis (Bt) and Bacillus popilliae are among the most successfulbiocontrol agents discovered to date. Insect pathogenicity has also beenattributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B.cereus. Microbial insecticides, particularly those obtained fromBacillus strains, have played an important role in agriculture asalternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance bygenetically engineering crop plants to produce pesticidal proteins fromBacillus. For example, corn and cotton plants have been geneticallyengineered to produce pesticidal proteins isolated from strains of Bt.These genetically engineered crops are now widely used in agricultureand have provided the farmer with an environmentally friendlyalternative to traditional insect-control methods. While they haveproven to be very successful commercially, these genetically engineered,insect-resistant crop plants provide resistance to only a narrow rangeof the economically important insect pests. In some cases, insects candevelop resistance to different insecticidal compounds, which raises theneed to identify alternative biological control agents for pest control.

Accordingly, there remains a need for new pesticidal proteins withdifferent ranges of insecticidal activity against insect pests, e.g.,insecticidal proteins which are active against a variety of insects inthe order Lepidoptera and the order Hemiptera including but not limitedto species belonging to the family Pentatomidae, the family Plataspidaeand the family Cydnidae. In addition, there remains a need forbiopesticides having activity against a variety of insect pests thathave developed resistance to existing pesticides.

SUMMARY OF THE INVENTION

Compositions and methods for conferring pesticidal activity to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for pesticidal andinsecticidal polypeptides, vectors comprising those nucleic acidmolecules, and host cells comprising the vectors. Compositions alsoinclude the pesticidal polypeptide sequences and antibodies to thosepolypeptides. The nucleic acid sequences can be used in DNA constructsor expression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant. Compositions also comprise transformed bacteria, plants,plant cells, tissues and seeds.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding PHI-4 polypeptides including amino acid substitutions,amino acid deletions, amino acid insertions, and fragments thereof, andcombinations thereof. Additionally, amino acid sequences correspondingto the PHI-4 polypeptides are encompassed. Nucleic acid sequences thatare complementary to a nucleic acid sequence of the embodiments, or thathybridize to a sequence of the embodiments are also encompassed.

Methods are provided for producing the polypeptides and for using thosepolypeptides for controlling, inhibiting growth or killing aLepidopteran, Coleopteran, nematode, fungi, Hemipteran and/or Dipteranpests. The transgenic plants of the embodiments express one or more ofthe pesticidal sequences disclosed herein. In various embodiments, thetransgenic plant further comprises one or more additional genes forinsect resistance, for example, one or more additional genes forcontrolling coleopteran, lepidopteran, hemipteran or nematode pests. Itwill be understood by one of skill in the art that the transgenic plantmay comprise any gene imparting an agronomic trait of interest.

Methods for detecting the nucleic acids and polypeptides of theembodiments in a sample are also included. A kit for detecting thepresence of a PHI-4 polypeptide or detecting the presence of anucleotide sequence encoding a PHI-4 polypeptide in a sample isprovided. The kit is provided along with all reagents and controlsamples necessary for carrying out a method for detecting the intendedagent, as well as instructions for use.

The compositions and methods of the embodiments are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. The compositions of the embodiments are alsouseful for generating altered or improved proteins that have pesticidalactivity, or for detecting the presence of PHI-4 polypeptides or nucleicacids in products or organisms.

The following embodiments are encompassed by the present disclosure.

Embodiment 1 is a PHI-4 polypeptide having improved insecticidalactivity compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 2 is the PHI-4 polypeptide of embodiment 1, wherein theinsecticidal activity is increased about 1.5 fold or greater compared toAXMI-205 (SEQ ID NO: 35).

Embodiment 3 is the PHI-4 polypeptide of embodiment 1, wherein theinsecticidal activity is increased about 2 fold or greater compared toAXMI-205 (SEQ ID NO: 35).

Embodiment 4 is the PHI-4 polypeptide of embodiment 1, wherein theinsecticidal activity is increased about 2.5 fold or greater compared toAXMI-205 (SEQ ID NO: 35).

Embodiment 5 is the PHI-4 polypeptide of embodiment 1, wherein theinsecticidal activity is increased about 3 fold or greater compared toAXMI-205 (SEQ ID NO: 35).

Embodiment 6 is the PHI-4 polypeptide of embodiment 1, wherein theinsecticidal activity is increased about 5 fold or greater compared toAXMI-205 (SEQ ID NO: 35).

Embodiment 7 is the PHI-4 polypeptide of any one of embodiments 1-6,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is against Western Corn Root Worm (WCRW) larvae.

Embodiment 8 is the PHI-4 polypeptide of any one of embodiments 1-7,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is quantified as a Mean FAE Index.

Embodiment 9 is the PHI-4 polypeptide of any one of embodiments 1-7,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is quantified as an EC50 value.

Embodiment 10 is the PHI-4 polypeptide of any one of embodiments 1-7,wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) isquantified as a Mean Deviation Score.

Embodiment 11 is the PHI-4 polypeptide of any one of embodiments 1-10,wherein the PHI-4 polypeptide comprises one or more amino acidsubstitutions compared to the native amino acid at position 40, 42, 43,46, 52, 97, 98, 99, 145, 150, 151, 153, 163, 171, 172, 182, 196, 206,210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342,346, 354, 355, 370, 389, 393, 396, 401, 402, 403, 410, 412, 416, 417,426, 442, 447, 452, 454, 455, 457, 461, 462, 500, 509, 520 or 527 of SEQID NO: 35.

Embodiment 12 is the PHI-4 polypeptide of embodiment 11, furthercomprising one or more amino acid substitutions at position 86, 359,464, 465, 466, 467, 468, 499 or 517.

Embodiment 13 is the PHI-4 polypeptide of embodiment 11 or 12, whereinthe amino acid at position 86 is Glu or Thr; the amino acid at position359 is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Aspor Asn; the amino acid at position 465 is Lys or Met, the amino acid atposition 467 is Val, Ala, Leu or Thr; the amino acid at position 468 isSer or Leu; the amino acid at position 499 is Glu or Ala, or the aminoacid at position 517 is Glu or Arg.

Embodiment 14 is the PHI-4 polypeptide of embodiment 11, 12 or 13,wherein the amino acid at position 40 is Leu or Ile; the amino acid atposition 42 is Asp or Asn; the amino acid at position 43 is Phe or Glu;the amino acid at position 46 is Glu or Asn; the amino acid at position52 is Ile or Val; the amino acid at position 97 is Arg, Asp, Glu or Asn;the amino acid at position 98 is Tyr or Phe; the amino acid at position99 is Lys or Leu; the amino acid at position 145 is Leu or Val; theamino acid at position 150 is Arg or Gin; the amino acid at position 151is Asp or Ser; the amino acid at position 153 is Leu or Ile; the aminoacid at position 163 is Leu or Val; the amino acid at position 171 isTyr or Phe; the amino acid at position 172 is Ile or Leu; the amino acidat position 182 is Asp or Gin; the amino acid at position 196 is Gin orAsn; the amino acid at position 206 is Tyr or Phe; the amino acid atposition 210 is Val or Ile; the amino acid at position 216 is Glu orGin; the amino acid at position 220 is Glu, Gin, His or Asp; the aminoacid at position 278 is Glu or Asn; the amino acid at position 283 isIle or Val; the amino acid at position 289 is Lys, Gin or Leu; the aminoacid at position 293 is Arg, Gin or Glu; the amino acid at position 328is Lys or Glu; the amino acid at position 333 is Ser, Lys or Val; theamino acid at position 334 is Gly, Lys or Arg; the amino acid atposition 336 is Gly or Ala; the amino acid at position 338 is Ser orVal; the amino acid at position 339 is Glu, Asn or Gin; the amino acidat position 342 is Ala or Ser; the amino acid at position 346 is Pro orAla; the amino acid at position 354 is Met or Leu; the amino acid atposition 355 is Val or Ile; the amino acid at position 370 is His orArg; the amino acid at position 389 is Trp or Leu; the amino acid atposition 393 is Trp or Leu; the amino acid at position 396 is Ala, Leu,Lys, Thr or Gly; the amino acid at position 401 is Ser, His, Gly, Lys orPro; the amino acid at position 402 is Lys, His, Gly or Trp; the aminoacid at position 403 is Asp or Tyr; the amino acid at position 410 isIle or Val; the amino acid at position 412 is Pro or Ala; the amino acidat position 416 is Arg or Glu; the amino acid at position 417 is Ala orSer; the amino acid at position 426 is Thr or Ser; the amino acid atposition 442 is Gin or Glu; the amino acid at position 447 is Asp orLys; the amino acid at position 452 is Gin or Lys; the amino acid atposition 454 is Arg or Gin; the amino acid at position 455 is Val orIle; the amino acid at position 457 is Trp or Asn; the amino acid atposition 461 is Thr or Ser; the amino acid at position 462 is Gly orAla; the amino acid at position 500 is Arg or Gin; the amino acid atposition 509 is Lys or Gin; the amino acid at position 520 is Lys, Gluor Gin; and the amino acid at position 527 is Gin or Lys.

Embodiment 15 is the PHI-4 polypeptide of any one of embodiments 1-11and 14, having 1 to 54 amino acid substitutions compared to SEQ ID NO:35.

Embodiment 16 is the PHI-4 polypeptide of any one of embodiments 1-11and 14, having 1 to 27 amino acid substitutions compared to SEQ ID NO:35.

Embodiment 17 is the PHI-4 polypeptide of any one of embodiments 1-11and 14, having 1 to 20 amino acid substitutions compared to SEQ ID NO:35.

Embodiment 18 is the PHI-4 polypeptide of any one of embodiments 1-11and 14, having 1 to 15 amino acid substitutions compared to SEQ ID NO:35.

Embodiment 19 is the PHI-4 polypeptide of any one of embodiments 12 or13, comprising 2 to 54 amino acid substitutions compared to SEQ ID NO:35.

Embodiment 20 is the PHI-4 polypeptide of any one of embodiments 12 or13, comprising 2 to 27 amino acid substitutions compared to SEQ ID NO:35.

Embodiment 21 is the PHI-4 polypeptide of any one of embodiments 12 or13, comprising 2 to 20 amino acid substitutions compared to SEQ ID NO:35.

Embodiment 22 is the PHI-4 polypeptide of any one of embodiments 12 or13, comprising 2 to 15 amino acid substitutions compared to SEQ ID NO:35.

Embodiment 23 is the PHI-4 polypeptide of any one of embodiments 1-22,wherein the PHI-4 polypeptide has at least 80% identity to SEQ ID NO:35.

Embodiment 24 is the PHI-4 polypeptide of any one of embodiments 1-22,wherein the PHI-4 polypeptide has at least 90% identity to SEQ ID NO:35.

Embodiment 25 is the PHI-4 polypeptide of any one of embodiments 1-22,wherein the PHI-4 polypeptide has at least 95% identity to SEQ ID NO:35.

Embodiment 26 is the PHI-4 polypeptide of any one of embodiments 1-22,wherein the PHI-4 polypeptide has at least 97% identity to SEQ ID NO:35.

Embodiment 27 is a PHI-4 polypeptide, comprising at least one amino acidsubstitution at a residue relative to SEQ ID NO: 35 in structural domainselected from:

-   -   a hydrophilic residue;    -   a residue in a membrane insertion initiation loop;    -   a residue in a receptor binding loop; and    -   a residue in a protease sensitive region,

wherein the PHI-4 polypeptide has increased insecticidal activitycompared to SEQ ID NO: 35.

Embodiment 28 is the PHI-4 polypeptide of embodiment 27, wherein thehydrophilic residues are Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp,Asn, Gin, and Cys.

Embodiment 29 is the PHI-4 polypeptide of embodiment 27 or 28, whereinthe membrane insertion loops are between about amino acid at position 92(Val) and 101 (Ala) and at position 211 (Gly) and 220 (Glu) relative toSEQ ID NO: 35.

Embodiment 30 is the PHI-4 polypeptide of embodiment 29, wherein themembrane insertion initiation loop residue is selected from position 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 211, 212, 213, 214, 215,216, 217, 218, 219, 220 of SEQ ID NO: 35.

Embodiment 31 is the PHI-4 polypeptide of any one of embodiments 27-30,wherein the receptor binding loops are between about amino acid 332(Asp) and 340 (Asp), 395 (Asp) and 403 (Asp), 458 (Asp) and 466 (Asp)relative to SEQ ID NO: 35.

Embodiment 32 is the PHI-4 polypeptide of embodiment 31, wherein thereceptor binding loop residue is selected from positions 332, 333, 334,335, 336, 337, 338, 339, 340, 395, 396, 397, 398, 399, 400, 401, 402,403, 458, 459, 460, 461, 462, 463, 464, 465, 466 of SEQ ID NO: 35.

Embodiment 33 is the PHI-4 polypeptide of any one of embodiments 27-32,wherein the protease sensitive region residue is selected from aboutamino acid residues between 305 (Lys) and 316 (Lys) and 500 (Arg) and535 (Lys) relative to SEQ ID NO: 35.

Embodiment 34 is the PHI-4 polypeptide of embodiment 27, wherein theprotease is trypsin.

Embodiment 35 is the PHI-4 polypeptide of any one of embodiments 27-34,wherein the PHI-4 polypeptide has at least 80% sequence identity to SEQID NO: 35.

Embodiment 36 is the PHI-4 polypeptide of any one of embodiments 27-34,wherein the PHI-4 polypeptide has at least 90% sequence identity to SEQID NO: 35.

Embodiment 37 is the PHI-4 polypeptide of any one of embodiments 27-34,wherein the PHI-4 polypeptide has at least 95% sequence identity to SEQID NO: 35.

Embodiment 38 is the PHI-4 polypeptide of any one of embodiments 27-34,wherein the PHI-4 polypeptide has at least 97% sequence identity to SEQID NO: 35.

Embodiment 39 is a PHI-4 polypeptide, comprising an amino acid sequenceof the formula,

(SEQ ID NO: 3)                5                   10                  15Met Xaa Ser Ala Ala Asn Ala Gly Xaa Leu Gly Asn Leu Xaa Gly                20                  25                  30Xaa Thr Ser Xaa Gly Met Xaa Tyr Xaa Val Asn Gly Leu Tyr Ala                35                  40                  45Ser Pro Glu Ser Leu Xaa Gly Gln Pro Leu Phe Xaa Xaa Gly Gly                50                  55                  60Xaa Leu Asp Ser Xaa Xaa Ile Glu Gly Xaa Xaa Xaa Xaa Phe Pro                65                  70                  75Xaa Ser Met His Val His Thr Tyr Phe His Ser Asp Xaa Xaa Gln                80                  85                  90Xaa Val Ser Xaa Xaa Ile Xaa Xaa Xaa Arg Xaa Xaa Xaa Ser Xaa                95                  100                 105His Val Gly Xaa Ser Gly Xaa Xaa Xaa Leu Phe Ser Xaa Ser Xaa                110                 115                 120Ser Val Asp Xaa Thr Thr Xaa Xaa Gln Gln Leu Xaa Glu Ile Thr                125                 130                 135Xaa Ser Ser Thr Arg Glu Xaa His Val Leu Trp Tyr Ile Ser Leu                140                 145                 150Pro Gly Ala Ala Thr Leu Xaa Ser Met Leu Xaa Xaa Xaa Xaa Xaa                155                 160                 165Xaa Asp Xaa Xaa Xaa Pro Asn Met Xaa Ala Met Xaa Leu Phe Xaa                170                 175                 180Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa Ala Ala Val Gly Gly Arg                185                 190                 195Leu Xaa Xaa Xaa Xaa Ala Ser Lys Xaa Leu Xaa Met Xaa Ser Ser                200                 205                 210Xaa Ser Leu Ser Thr Thr Xaa Xaa Xaa Ser Xaa Xaa Ala Xaa Xaa                215                 220                 225Gly Glu Ile Xaa Ile Xaa His Gly Ser Xaa Met Glu Lys Gln Val                230                 235                 240Asn Ser Phe Xaa Xaa Xaa Ser Thr Ile Arg Xaa Thr Ala Thr Gly                245                 250                 255Gly Lys Pro Gly Xaa Thr Xaa Arg Ile Leu His Gly Pro Asp Ser                260                 265                 270Xaa Xaa Ala Phe Ser Xaa Trp Ala Xaa Ser Leu Leu Xaa Tyr Ala                275                 280                 285Thr Leu Met Asp Phe Xaa Thr Xaa Ser Leu Xaa Xaa Ile Xaa Ala                290                 295                 300Leu Xaa Asp Xaa Pro Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Ala Xaa                305                 310                 315Pro Xaa Xaa Met Xaa Xaa Ser Gln Xaa Ser Ile Pro Xaa Val Asp                320                 325                 330Xaa Val Leu Leu Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly                335                 340                 345Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa                350                 355                 360Xaa Ser Thr Ser Xaa Xaa Tyr Lys Xaa Xaa Gly Gln Phe Xaa Gln                365                 370                 375Arg Xaa His Xaa Ser Val Ala Asp Gly His Xaa Pro Ile Xaa Xaa                380                 385                 390Asp Leu Phe Asp Xaa Gly Xaa Xaa Xaa Xaa Pro Val Gly Xaa Gln                395                 400                 405Xaa Val Trp Asp Xaa Xaa Xaa Xaa Gly Lys Xaa Xaa Xaa Tyr Xaa                410                 415                 420Cys Trp Arg Xaa Xaa Xaa Xaa Gln Gly Tyr Xaa Xaa Xaa Gly Asp                425                 430                 435Val Xaa Met Leu Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro                440                 445                 450Xaa Xaa Xaa Cys Xaa His Xaa Ser Leu Xaa Ala Xaa Xaa Xaa Thr                455                 460                 465Leu Xaa Xaa Xaa Xaa Trp Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa                470                475                 480Xaa Val Ser Leu Trp Xaa Pro Gly Ala Ala Gly Ala Val Ala Ser                485                 490                 495Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser                500                 505                 510Gly Xaa Ile Xaa Xaa Leu Xaa Gly Ser Ile Ala Cys Val Xaa Thr                515                 520                 525Ser Ala Ile Ala Ser Met Xaa Xaa Met Xaa Ser Met Leu Ser Xaa                530                 535His Xaa Gly Met Glu Ala Met Met Ser Lys Leu,wherein Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lysor Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val orAsp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser;Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met;Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu;Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie orVal; Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys;Xaa at position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; Xaaat position 58 is Thr or Ser; Xaa at position 61 is Arg, Lys or Glu; Xaaat position 73 is Phe or Tyr; Xaa at position 74 is Lys, Glu, Gly, Arg,Met, Leu, His or Asp; Xaa at position 76 is Asp or Gin; Xaa at position79 is Lys or Glu; Xaa at position 80 is Glu or Ser; Xaa at position 82is Glu, lie, Leu, Tyr or Gin; Xaa at position 83 is Glu or Gin; Xaa atposition 84 is Tyr or Phe; Xaa at position 86 is Glu or Gin; Xaa atposition 87 is Lys or Gin; Xaa at position 88 is Met, lie or Leu; Xaa atposition 90 is Gin or Glu; Xaa at position 94 is Val or lie; Xaa atposition 97 is Arg, Asn, Asp, Glu, Gin, Gly or Ser; Xaa at position 98is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie, Met, Phe, Cys,Val or Asn; Xaa at position 103 is Ala or Gly; Xaa at position 105 isLeu or lie; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn,Glu, Cys, Ala or Arg; Xaa at position 112 is Thr or Ser; Xaa at position113 is Asp, Glu or Met; Xaa at position 117 is Thr or Ser; Xaa atposition 121 is Tyr or Phe; Xaa at position 127 is Ala or Thr; Xaa atposition 142 is Arg or Glu; Xaa at position 146 is Arg or Gin; Xaa atposition 147 is Arg, Glu or Gin; Xaa at position 148 is Asp, Phe, Pro,Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gin or Gly; Xaa at position 149is Phe or Val; Xaa at position 150 is Arg, Gin or Glu; Xaa at position151 is Asp, Ser, Ala, Asn, Trp, Val, Gin, Cys, Met, Leu, Arg or Glu; Xaaat position 153 is Leu or lie; Xaa at position 154 is Asn or Asp; Xaa atposition 155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa atposition 162 is Glu, Asp or Gin; Xaa at position 165 is Lys, Glu, Gin,Pro, Thr, Ala, Leu, Gly, Asp, Val, His, lie, Met, Trp, Phe, Tyr or Arg;Xaa at position 166 is Arg or Gin; Xaa at position 167 is Tyr, Trp orCys; Xaa at position 170 is Tyr or His; Xaa at position 171 is Tyr orPhe; Xaa at position 172 is lie, Leu or Val; Xaa at position 173 is Seror Ala; Xaa at position 174 is Glu or Gin; Xaa at position 182 is Asp orGin; Xaa at position 183 is Tyr or Val; Xaa at position 184 is Ser orThr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is Thr, Lysor lie; Xaa at position 191 is Lys or Gin; Xaa at position 193 is Asp orAsn; Xaa at position 196 is Gin, Lys, Asn, Asp, Glu, Ala, lie or Arg;Xaa at position 202 is Ala or Val; Xaa at position 203 is Glu, Thr orHis; Xaa at position 204 is Met or Ala; Xaa at position 206 is Tyr orPhe; Xaa at position 207 is Lys or Gin; Xaa at position 209 is Leu orPro; Xaa at position 210 is Val or lie; Xaa at position 214 is Lys, Seror Gin; Xaa at position 216 is Glu, Gin, Phe, Val, Tyr or Arg; Xaa atposition 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, Gin, Arg, Trp, Met,Ala, Phe, lie, Leu, Cys or Asn; Xaa at position 229 is Arg or Glu; Xaaat position 230 is Ser or Glu; Xaa at position 231 is Asn or Ser; Xaa atposition 236 is Leu or Pro; Xaa at position 245 is Met or Leu; Xaa atposition 247 is Asp or Tyr; Xaa at position 256 is Gin, Lys or Glu; Xaaat position 257 is Gin, lie, Glu, Cys, Ser, His, Trp or Met; Xaa atposition 261 is Gin, Glu or Lys; Xaa at position 264 is Glu or Gin; Xaaat position 268 is Asp or Asn; Xaa at position 276 is Ser or Ala; Xaa atposition 278 is Glu, Asn or Gin; Xaa at position 281 is Gin, Lys or Glu;Xaa at position 282 is Pro or Gly; Xaa at position 284 is Trp or Arg;Xaa at position 287 is Ala or Cys; Xaa at position 289 is Lys, Leu, Val,Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn,Cys, Gly or Ala; Xaa at position 291 is Glu or Gin; Xaa at position 292is Arg or Gin; Xaa at position 293 is Arg, Glu or Gin; Xaa at position294 is Val or Ala; Xaa at position 296 is Leu or lie; Xaa at position297 is Glu or Gin; Xaa at position 298 is Asp or Gin; Xaa at position300 is Phe or Tyr; Xaa at position 302 is Glu or Gin; Xaa at position303 is Phe or Tyr; Xaa at position 305 is Lys or Gin; Xaa at position306 is Gin or Lys; Xaa at position 309 is Gin, Lys or Glu; Xaa atposition 313 is Lys, Gin or Arg; Xaa at position 316 is Lys or Gin; Xaaat position 328 is Lys, Glu or Gin; Xaa at position 331 is Glu, Asn orGin; Xaa at position 333 is Ser, Arg, Gly, Lys, Val, Asn, Ala, His, Gin,Thr, Asp, lie, Leu, Cys or Glu; Xaa at position 334 is Gly, Arg, Lys,lie or Trp; Xaa at position 335 is Ser or Ala; Xaa at position 336 isGly or Ala; Xaa at position 337 is Ala, Val or Gly; Xaa at position 338is Ser, His, Val, Lys, Ala, Gly, Thr, lie, Glu, Met, Arg, Pro, Asp, Asnor Leu; Xaa at position 339 is Glu, Asn, Gin, lie, Pro, Met, Ser, Ala,Cys, Phe, Val, Leu, Asp, Trp, His or Arg; Xaa at position 341 is Leu orVal; Xaa at position 342 is Ala, Ser or Val; Xaa at position 343 is Valor lie; Xaa at position 344 is Phe or Trp; Xaa at position 345 is Asn orHis; Xaa at position 346 is Pro or Ala; Xaa at position 350 is Asn orSer; Xaa at position 351 is Gly or Val; Xaa at position 354 is Met orLeu; Xaa at position 355 is Val, lie or Leu; Xaa at position 359 is Glyor Ala; Xaa at position 362 is Asn or Ser; Xaa at position 364 is Ala orSer; Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is Pheor lie; Xaa at position 375 is Lys or Arg; Xaa at position 380 is Leu orGly; Xaa at position 382 is Val, Asp or Leu; Xaa at position 383 is Leu,lie or Val; Xaa at position 384 is Lys, Ala or Gly; Xaa at position 385is Ala or Gly; Xaa at position 389 is Trp or Tyr; Xaa at position 391 isArg, Leu, Glu, Gin or Asp; Xaa at position 395 is Asp or Cys; Xaa atposition 396 is Ala, Leu, Lys, Asn, Gly, lie, Met, Arg, Tyr, Gin or His;Xaa at position 397 is Gly, Arg or Ala; Xaa at position 398 is Ser, Ginor Cys; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val, Arg, lie,Asn, Phe, Thr, Ala, Asp, Met, Gin or Glu; Xaa at position 402 is Lys,Phe, His, Arg, Trp, Gly, Asn, Leu, Tyr, Thr, Val, Met, Pro or Ala; Xaaat position 403 is Asp, Tyr, Trp, Phe or Glu; Xaa at position 405 is Alaor Ser; Xaa at position 409 is Ala or Pro; Xaa at position 410 is lie orVal; Xaa at position 411 is Pro or Ala; Xaa at position 412 is Pro orAla; Xaa at position 416 is Arg, Glu or Gin; Xaa at position 417 is Ala,Ser or Cys; Xaa at position 418 is Leu or Met; Xaa at position 422 isMet or Val; Xaa at position 426 is Thr or Ser; Xaa at position 436 isAsp or Lys; Xaa at position 437 is Tyr or Val; Xaa at position 438 isVal or Arg; Xaa at position 440 is Val or Leu; Xaa at position 442 isGin, Lys or Glu; Xaa at position 445 is Cys, Leu or Thr; Xaa at position447 is Asp, Lys, Tyr, Ser, Glu, lie, Gly, Pro, Leu, Phe, Trp or Thr; Xaaat position 448 is Val or Ala; Xaa at position 449 is Gin or Glu; Xaa atposition 452 is Gin, Lys or Glu; Xaa at position 453 is Asn or Asp; Xaaat position 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp, Gin,Gly, His, Asp, Leu, Thr, Pro or Asn; Xaa at position 455 is Val or lie;Xaa at position 457 is Trp or Asn; Xaa at position 459 is Lys, Met, Val,Trp, Gin, lie, Thr, Ser, His, Cys, Tyr, Pro, Asn, Ala, Arg or Glu; Xaaat position 460 is Gly or Ala; Xaa at position 461 is Thr or Ser; Xaa atposition 462 is Gly or Ala; Xaa at position 463 is Ala, Ser or Gly; Xaaat position 464 is Arg, Gly, His, Gin, Thr or Phe; Xaa at position 465is Lys, Asn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu;Xaa at position 466 is Asp or Arg; Xaa at position 471 is Gin, Lys orGlu; Xaa at position 497 is Asp or Gin; Xaa at position 499 is Glu orGin; Xaa at position 500 is Arg, Gin or Lys; Xaa at position 502 is Arg,Glu or Gin; Xaa at position 509 is Lys, Gin or Glu; Xaa at position 517is Gin, Cys, Asn, Val or Pro; Xaa at position 518 is Glu or Gin; Xaa atposition 520 is Lys, Gin or Glu; Xaa at position 525 is Gin or Lys; andXaa at position 527 is Gin, Lys, Pro, Cys, Glu, Ser, His, Phe or Trp;wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 3 isan amino acid different from the corresponding amino acid of SEQ ID NO:35 and wherein the PHI-4 polypeptide has increased insecticidal activitycompared to SEQ ID NO: 35.

Embodiment 40 is a PHI-4 polypeptide, comprising an amino acid sequenceof the formula

(SEQ ID NO: 4)                5                   10                  15Met Xaa Ser Ala Ala Asn Ala Gly Gln Leu Gly Asn Leu Pro Gly                20                  25                  30Val Thr Ser Met Gly Met Gly Tyr Xaa Val Asn Gly Leu Tyr Ala                35                  40                  45Ser Pro Glu Ser Leu Leu Gly Gln Pro Leu Phe Xaa Xaa Gly Gly                50                  55                  60Xaa Leu Asp Ser Ile Glu Ile Glu Gly Arg Ser Tyr Thr Phe Pro                65                  70                  75Arg Ser Met His Val His Thr Tyr Phe His Ser Asp Phe Xaa Gln                80                  85                  90Asp Val Ser Xaa Glu Ile Xaa Glu Tyr Arg Glu Lys Met Ser Gln                95                  100                 105His Val Gly Val Ser Gly Xaa Xaa Xaa Leu Phe Ser Ala Ser Leu                110                 115                 120Ser Val Asp Xaa Thr Thr Thr Asp Gln Gln Leu Thr Glu Ile Thr                125                 130                 135Tyr Ser Ser Thr Arg Glu Ala His Val Leu Trp Tyr Ile Ser Leu                140                 145                 150Pro Gly Ala Ala Thr Leu Arg Ser Met Leu Arg Xaa Xaa Phe Xaa                155                 160                 165Xaa Asp Xaa Asn Asn Pro Asn Met Pro Ala Met Xaa Leu Phe Xaa                170                 175                 180Xaa Tyr Gly Pro Tyr Xaa Ile Ser Xaa Ala Ala Val Gly Gly Arg                185                 190                 195Leu Xaa Tyr Ser Ala Ala Ser Lys Thr Leu Lys Met Asp Ser Ser                200                 205                 210Xaa Ser Leu Ser Thr Thr Ala Xaa Met Ser Xaa Lys Ala Leu Val                215                 220                 225Gly Glu Ile Lys Ile Xaa His Gly Ser Xaa Met Glu Lys Gln Val                230                 235                 240Asn Ser Phe Arg Ser Asn Ser Thr Ile Arg Leu Thr Ala Thr Gly                245                 250                 255Gly Lys Pro Gly Met Thr Xaa Arg Ile Leu His Gly Pro Asp Ser                260                 265                 270Xaa Xaa Ala Phe Ser Xaa Trp Ala Glu Ser Leu Leu Asp Tyr Ala                275                 280                 285Thr Leu Met Asp Phe Ser Thr Xaa Ser Leu Xaa Pro Ile Trp Ala                290                 295                 300Leu Ala Asp Xaa Pro Glu Arg Xaa Val Glu Leu Glu Asp Ala Phe                305                 310                 315Pro Glu Phe Met Lys Gln Ser Gln Gln Ser Ile Pro Xaa Val Asp                320                 325                 330Lys Val Leu Leu Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly                335                 340                 345Glu Asp Xaa Xaa Ser Xaa Ala Xaa Xaa Asp Leu Ala Xaa Phe Asn                350                 355                 360Xaa Ser Thr Ser Asn Gly Tyr Lys Met Xaa Gly Gln Phe Xaa Gln                365                 370                 375Arg Asn His Ala Ser Val Ala Asp Gly His Ala Pro Ile Phe Lys                380                 385                 390Asp Leu Phe Asp Leu Gly Val Leu Lys Ala Pro Val Gly Trp Gln                395                 400                 405Xaa Val Trp Asp Asp Xaa Gly Ser Gly Lys Xaa Xaa Xaa Tyr Ala                410                 415                 420Cys Trp Arg Ala Ile Xaa Xaa Gln Gly Tyr Xaa Xaa Xaa Gly Asp                425                 430                 435Val Met Met Leu Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro                440                 445                 450Asp Tyr Val Cys Xaa His Gln Ser Leu Cys Ala Xaa Val Gln Thr                455                 460                 465Leu Xaa Asn Xaa Xaa Trp Trp Asp Xaa Gly Xaa Xaa Xaa Xaa Xaa                470                 475                 480Asp Val Ser Leu Trp Xaa Pro Gly Ala Ala Gly Ala Val Ala Ser                485                 490                 495Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser                500                 505                 510Gly Asp Ile Glu Xaa Leu Arg Gly Ser Ile Ala Cys Val Xaa Thr                515                 520                 525Ser Ala Ile Ala Ser Met Gln Glu Met Xaa Ser Met Leu Ser Gln                530                 535His Xaa Gly Met Glu Ala Met Met Ser Lys Leu,wherein Xaa at position 2 is Ala or Arg; Xaa at position 24 is Asp orAsn; Xaa at position 42 is Asp or Asn; Xaa at position 43 is Phe or Glu;Xaa at position 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly;Xaa at position 79 is Lys or Glu; Xaa at position 82 is Glu, lie, Leu orTyr; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin or Gly; Xaa atposition 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie orMet; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa atposition 147 is Arg or Glu; Xaa at position 148 is Asp, Phe or Pro; Xaaat position 150 is Arg or Gin; Xaa at position 151 is Asp, Ser, Ala orAsn; Xaa at position 153 is Leu or lie; Xaa at position 162 is Glu orGin; Xaa at position 165 is Lys, Glu or Gin; Xaa at position 166 is Argor Gin; Xaa at position 171 is Tyr or Phe; Xaa at position 174 is Glu orGin; Xaa at position 182 is Asp or Gin; Xaa at position 196 is Gin, Lys,Asn or Asp; Xaa at position 203 is Glu, Thr or His; Xaa at position 206is Tyr or Phe; Xaa at position 216 is Glu or Gin; Xaa at position 220 isGlu, His, Asp, Thr, Tyr, Val, Ser or Gin; Xaa at position 247 is Asp orTyr; Xaa at position 256 is Gin or Lys; Xaa at position 257 is Gin orlie; Xaa at position 261 is Gin or Glu; Xaa at position 278 is Glu orAsn; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 289 is Lys,Leu, Val, Pro, Glu, Gin, Tyr, Thr or Asp; Xaa at position 293 is Arg,Glu or Gin; Xaa at position 313 is Lys or Gin; Xaa at position 328 isLys, Glu or Gin; Xaa at position 333 is Ser, Gly, Lys, Val or Asn; Xaaat position 334 is Gly, Arg, Lys or lie; Xaa at position 336 is Gly orAla; Xaa at position 338 is Ser, His, Val, Lys or Ala; Xaa at position339 is Glu, Asn, lie or Pro; Xaa at position 343 is Val or lie; Xaa atposition 346 is Pro or Ala; Xaa at position 355 is Val or lie; Xaa atposition 359 is Gly or Ala; Xaa at position 391 is Arg, Glu or Gin; Xaaat position 396 is Ala, Leu, Lys, Asn or Gly; Xaa at position 401 isSer, His, Pro, Gly, Lys, Val or Arg; Xaa at position 402 is Lys, Phe,His, Arg, Gly, Trp, Thr, Asn, Tyr or Met; Xaa at position 403 is Asp orTyr; Xaa at position 411 is Pro or Ala; Xaa at position 412 is Pro orAla; Xaa at position 416 is Arg or Glu; Xaa at position 417 is Ala orSer; Xaa at position 418 is Leu or Met; Xaa at position 426 is Thr orSer; Xaa at position 440 is Val or Leu; Xaa at position 447 is Asp, Lys,Tyr, Ser, Glu or lie; Xaa at position 452 is Gin, Lys or Glu; Xaa atposition 454 is Arg, Tyr, Met, Ser, Val, lie, Lys, Phe, Trp or Gin; Xaaat position 455 is Val or lie; Xaa at position 459 is Lys, Met, Val,Trp, Gin, lie or Tyr; Xaa at position 461 is Thr or Ser; Xaa at position462 is Gly or Ala; Xaa at position 463 is Ala or Ser; Xaa at position464 is Arg, Gly or His; Xaa at position 465 is Lys, Asn, Val, Met, Pro,Gly or Arg; Xaa at position 471 is Gin, Lys or Glu; Xaa at position 500is Arg or Gin; Xaa at position 509 is Lys or Gin; Xaa at position 520 isLys, Gin or Glu; and Xaa at position 527 is Gin, Lys, Pro, Cys or Glu;wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 4 isan amino acid different from the corresponding amino acid of SEQ ID NO:35 and wherein the PHI-4 polypeptide has increased insecticidal activitycompared to SEQ ID NO: 35.

Embodiment 41 is the PHI-4 polypeptide of embodiment 39 or 40, furthercomprising one or more amino acid substitutions at position 86, 359,464, 465, 466, 467, 468, 499 or 517 of SEQ ID NO: 3 or SEQ ID NO: 4.

Embodiment 42 is the PHI-4 polypeptide of embodiment 41, wherein theamino acid at position 86 is Glu or Thr; the amino acid at position 359is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp orAsn; the amino acid at position 465 is Lys or Met, the amino acid atposition 467 is Val, Ala, Leu or Thr; the amino acid at position 468 isSer or Leu; the amino acid at position 499 is Glu or Ala, or the aminoacid at position 517 is Glu or Arg.

Embodiment 43 is the PHI-4 polypeptide of embodiment 39-42, furthercomprising one or more conservative amino acid substitution, insertionof one or more amino acids, deletion of one or more amino acids, andcombinations thereof.

Embodiment 44 is the PHI-4 polypeptide of any one of embodiments 39-43,wherein the insecticidal activity is increased about 1.5 fold or greatercompared to AXMI-205 (SEQ ID NO: 35).

Embodiment 45 is the PHI-4 polypeptide of any one of embodiments 39-43,wherein the insecticidal activity is increased about 2 fold or greatercompared to AXMI-205 (SEQ ID NO: 35).

Embodiment 46 is the PHI-4 polypeptide of any one of embodiments 39-43,wherein the insecticidal activity is increased about 2.5 fold or greatercompared to AXMI-205 (SEQ ID NO: 35).

Embodiment 47 is the PHI-4 polypeptide of any one of embodiments 39-43,wherein the insecticidal activity is increased about 3 fold or greatercompared to AXMI-205 (SEQ ID NO: 35).

Embodiment 48 is the PHI-4 polypeptide of any one of embodiments 39-43,wherein the insecticidal activity is increased about 5 fold or greatercompared to AXMI-205 (SEQ ID NO: 35).

Embodiment 49 is the PHI-4 polypeptide of any one of embodiments 39-48,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is against Western Corn Root Worm (WCRW) larvae.

Embodiment 50 is the PHI-4 polypeptide of any one of embodiments 39-49,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is quantified as a Mean FAE Index.

Embodiment 51 is the PHI-4 polypeptide of any one of embodiments 39-49,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is quantified as an EC50 value.

Embodiment 52 is the PHI-4 polypeptide of any one of embodiments 39-49,wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) isquantified as a Mean Deviation Score.

Embodiment 53 is the PHI-4 polypeptide of any one of embodiments 39-49,having 1 to 54 amino acid substitutions at a position(s) designated asXaa in SEQ ID NO: 3 or 4.

Embodiment 54 is the PHI-4 polypeptide of any one of embodiments 39-49,having 1 to 27 amino acid substitutions at a position(s) designated asXaa in SEQ ID NO: 3 or 4.

Embodiment 55 is the PHI-4 polypeptide of any one of embodiments 39-49,having 1 to 20 amino acid substitutions at a position(s) designated asXaa in SEQ ID NO: 3 or 4.

Embodiment 56 is the PHI-4 polypeptide of any one of embodiments 39-49,having 1 to 15 amino acid substitutions at a position(s) designated asXaa in SEQ ID NO: 3 or 4.

Embodiment 57 is the PHI-4 polypeptide of any one of embodiments 1-56,wherein 1-25 amino acids are deleted from the N-terminus of the PHI-4polypeptide and/or C-terminus of the PHI-4 polypeptide.

Embodiment 58 is the PHI-4 polypeptide of any one of embodiments 1-53,wherein 1-20 amino acids are deleted from the C-terminus of the PHI-4polypeptide.

Embodiment 59 is a polynucleotide encoding a PHI-4 polypeptide, whereinthe PHI-4 polypeptide has improved insecticidal activity compared toAXMI-205 (SEQ ID NO: 35).

Embodiment 60 is the polynucleotide of embodiment 59, wherein theinsecticidal activity of the PHI-4 polypeptide is increased about 1.5fold or greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 61 is the polynucleotide of embodiment 59, wherein theinsecticidal activity of the PHI-4 polypeptide is increased about 2 foldor greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 62 is the polynucleotide of embodiment 59, wherein theinsecticidal activity of the PHI-4 polypeptide is increased about 2.5fold or greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 63 is the polynucleotide of embodiment 59, wherein theinsecticidal activity of the PHI-4 polypeptide is increased about 3 foldor greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 64 is the polynucleotide of embodiment 59, wherein theinsecticidal activity of the PHI-4 polypeptide is increased about 5 foldor greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 65 is the polynucleotide of any one of embodiments 59-64,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is against Western Corn Root Worm (WCRW) larvae.

Embodiment 66 is the polynucleotide of any one of embodiments 59-64,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is quantified as a Mean FAE Index.

Embodiment 67 is the polynucleotide of any one of embodiments 59-64,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is quantified as an EC50 value.

Embodiment 68 is the polynucleotide of any one of embodiments 59-64,wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) isquantified as a Mean Deviation Score.

Embodiment 69 is the polynucleotide of any one of embodiments 59-68,wherein the PHI-4 polypeptide comprises one or more amino acidsubstitutions compared to the native amino acid at position 40, 42, 43,46, 52, 97, 98, 99, 145, 150, 151, 153, 163, 171, 172, 182, 196, 206,210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342,346, 354, 355, 370, 389, 393, 396, 401, 402, 403, 410, 412, 416, 417,426, 442, 447, 452, 454, 455, 457, 461, 462, 500, 509, 520 or 527 of SEQID NO: 35.

Embodiment 70 is the polynucleotide of embodiment 69, wherein the PHI-4polypeptide further comprises one or more amino acid substitutionscompared to the native amino acid at position 86, 359, 464, 465, 466,467, 468, 499 or 517 of SEQ ID NO: 35.

Embodiment 71 is the polynucleotide of embodiment 70, wherein the aminoacid at position 86 is Glu or Thr; the amino acid at position 359 is Glyor Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; theamino acid at position 465 is Lys or Met, the amino acid at position 467is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu;the amino acid at position 499 is Glu or Ala, or the amino acid atposition 517 is Glu or Arg.

Embodiment 72 is the polynucleotide of any one of embodiments 69-71,wherein the amino acid at position 40 is Leu or lie; the amino acid atposition 42 is Asp or Asn; the amino acid at position 43 is Phe or Glu;the amino acid at position 46 is Glu or Asn; the amino acid at position52 is lie or Val; the amino acid at position 97 is Arg, Asp, Glu or Asn;the amino acid at position 98 is Tyr or Phe; the amino acid at position99 is Lys or Leu; the amino acid at position 145 is Leu or Val; theamino acid at position 150 is Arg or Gin; the amino acid at position 151is Asp or Ser; the amino acid at position 153 is Leu or lie; the aminoacid at position 163 is Leu or Val; the amino acid at position 171 isTyr or Phe; the amino acid at position 172 is lie or Leu; the amino acidat position 182 is Asp or Gin; the amino acid at position 196 is Gin orAsn; the amino acid at position 206 is Tyr or Phe; the amino acid atposition 210 is Val or lie; the amino acid at position 216 is Glu orGin; the amino acid at position 220 is Glu, Gin, His or Asp; the aminoacid at position 278 is Glu or Asn; the amino acid at position 283 islie or Val; the amino acid at position 289 is Lys, Gin or Leu; the aminoacid at position 293 is Arg, Gin or Glu; the amino acid at position 328is Lys or Glu; the amino acid at position 333 is Ser, Lys or Val; theamino acid at position 334 is Gly, Lys or Arg; the amino acid atposition 336 is Gly or Ala; the amino acid at position 338 is Ser orVal; the amino acid at position 339 is Glu, Asn or Gin; the amino acidat position 342 is Ala or Ser; the amino acid at position 346 is Pro orAla; the amino acid at position 354 is Met or Leu; the amino acid atposition 355 is Val or lie; the amino acid at position 370 is His orArg; the amino acid at position 389 is Trp or Leu; the amino acid atposition 393 is Trp or Leu; the amino acid at position 396 is Ala, Leu,Lys, Thr or Gly; the amino acid at position 401 is Ser, His, Gly, Lys orPro; the amino acid at position 402 is Lys, His, Gly or Trp; the aminoacid at position 403 is Asp or Tyr; the amino acid at position 410 islie or Val; the amino acid at position 412 is Pro or Ala; the amino acidat position 416 is Arg or Glu; the amino acid at position 417 is Ala orSer; the amino acid at position 426 is Thr or Ser; the amino acid atposition 442 is Gin or Glu; the amino acid at position 447 is Asp orLys; the amino acid at position 452 is Gin or Lys; the amino acid atposition 454 is Arg or Gin; the amino acid at position 455 is Val orlie; the amino acid at position 457 is Trp or Asn; the amino acid atposition 461 is Thr or Ser; the amino acid at position 462 is Gly orAla; the amino acid at position 500 is Arg or Gin; the amino acid atposition 509 is Lys or Gin; the amino acid at position 520 is Lys, Gluor Gin; and the amino acid at position 527 is Gin or Lys.

Embodiment 73 is the polynucleotide of any one of embodiments 59-69 and72, wherein the PHI-4 polypeptide has 1 to 54 amino acid substitutionscompared to SEQ ID NO: 35.

Embodiment 74 is the polynucleotide of any one of embodiments 59-69 and72, wherein the PHI-4 polypeptide has 1 to 27 amino acid substitutionscompared to SEQ ID NO: 2.

Embodiment 75 is the polynucleotide of any one of embodiments 59-69 and72, wherein the PHI-4 polypeptide has 1 to 20 amino acid substitutionscompared to SEQ ID NO: 35.

Embodiment 76 is the polynucleotide of any one of embodiments 59-69 and72, wherein the PHI-4 polypeptide has 1 to 15 amino acid substitutionscompared to SEQ ID NO: 35.

Embodiment 77 is the polynucleotide of embodiments 70 or 71, wherein thePHI-4 polypeptide has 2 to 54 amino acid substitutions compared to SEQID NO: 35.

Embodiment 78 is the polynucleotide of embodiments 70 or 71, wherein thePHI-4 polypeptide has 2 to 27 amino acid substitutions compared to SEQID NO: 35.

Embodiment 79 is the polynucleotide of embodiments 70 or 71, wherein thePHI-4 polypeptide has 2 to 20 amino acid substitutions compared to SEQID NO: 35.

Embodiment 80 is the polynucleotide of embodiments 70 or 71, wherein thePHI-4 polypeptide has 2 to 15 amino acid substitutions compared to SEQID NO: 35.

Embodiment 81 is the polynucleotide of any one of embodiments 59-80,wherein the PHI-4 polypeptide has at least 80% identity to SEQ ID NO:35.

Embodiment 82 is the polynucleotide of any one of embodiments 59-80,wherein the PHI-4 polypeptide has at least 90% identity to SEQ ID NO:35.

Embodiment 83 is the polynucleotide of any one of embodiments 59-80,wherein the PHI-4 polypeptide has at least 95% identity to SEQ ID NO:35.

Embodiment 84 is the polynucleotide of any one of embodiments 59-80,wherein the PHI-4 polypeptide has at least 97% identity to SEQ ID NO:35.

Embodiment 85 is a polynucleotide encoding a PHI-4 polypeptide, whereinthe PHI-4 polypeptide has at least one amino acid substitution at aresidue relative to SEQ ID NO: 35 in structural domain selected from:

-   -   a hydrophilic residue;    -   a residue in a membrane insertion initiation loop;    -   a residue in a receptor binding loop; and    -   a residue in a protease sensitive region,

wherein the PHI-4 polypeptide has increased insecticidal activitycompared to SEQ ID NO: 35.

Embodiment 86 is the polynucleotide of embodiment 85, wherein thehydrophilic residues are Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp,Asn, Gin, and Cys. Embodiment 87 is the polynucleotide of embodiment 85or 86, wherein the membrane insertion loops are between about amino acidat position 92 (Val) and 101 (Ala) and at position 211 (Gly) and 220(Glu) relative to SEQ ID NO: 35.

Embodiment 88 is the polynucleotide of embodiment 87, wherein the PHI-4polypeptide has one or more amino substitution compared to the nativeamino acid at position 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 211, 212, 213, 214, 215, 216, 217, 218, 219, and 220 of SEQ ID NO:35.

Embodiment 89 is the polynucleotide of any one of embodiments 85, 86, 87or 88, wherein the receptor binding loops are between about amino acid332 (Asp) and 340 (Asp), 395 (Asp) and 403 (Asp), 458 (Asp) and 466(Asp) relative to SEQ ID NO: 35.

Embodiment 90 is the polynucleotide of embodiment 89, wherein the PHI-4polypeptide has one or more amino substitution compared to the nativeamino acid at position 332, 333, 334, 335, 336, 337, 338, 339, 340, 395,396, 397, 398, 399, 400, 401, 402, 403, 458, 459, 460, 461, 462, 463,464, 465, 466 of SEQ ID NO: 35.

Embodiment 91 is the polynucleotide of any one of embodiments 85, 86,87, 88, 89 or 90, wherein the protease sensitive region residue isselected from about amino acid residues between position 305 (Lys) andposition 316 (Lys) and/or position 500 (Arg) and position 535 (Lys)relative to SEQ ID NO: 35.

Embodiment 92 is the polynucleotide of any one of embodiments 85, 86,87, 88, 89, 90 or 91, wherein the protease is trypsin.

Embodiment 93 is the polynucleotide of any one of embodiments 85-92,wherein the PHI-4 polypeptide has at least 80% sequence identity to SEQID NO: 35.

Embodiment 94 is the polynucleotide of any one of embodiments 85-92,wherein the PHI-4 polypeptide has at least 90% sequence identity to SEQID NO: 35.

Embodiment 95 is the polynucleotide of any one of embodiments 85-92,wherein the PHI-4 polypeptide has at least 95% sequence identity to SEQID NO: 35.

Embodiment 96 is the polynucleotide of any one of embodiments 85-92,wherein the PHI-4 polypeptide has at least 97% sequence identity to SEQID NO: 35.

Embodiment 97 is a polynucleotide encoding a PHI-4 polypeptide, whereinthe PHI-4 polypeptide comprises an amino acid sequence of the formula,

(SEQ ID NO: 3)                5                   10                  15Met Xaa Ser Ala Ala Asn Ala Gly Xaa Leu Gly Asn Leu Xaa Gly                20                  25                  30Xaa Thr Ser Xaa Gly Met Xaa Tyr Xaa Val Asn Gly Leu Tyr Ala                35                  40                  45Ser Pro Glu Ser Leu Xaa Gly Gln Pro Leu Phe Xaa Xaa Gly Gly                50                  55                  60Xaa Leu Asp Ser Xaa Xaa Ile Glu Gly Xaa Xaa Xaa Xaa Phe Pro                65                  70                  75Xaa Ser Met His Val His Thr Tyr Phe His Ser Asp Xaa Xaa Gln                80                  85                  90Xaa Val Ser Xaa Xaa Ile Xaa Xaa Xaa Arg Xaa Xaa Xaa Ser Xaa                95                  100                 105His Val Gly Xaa Ser Gly Xaa Xaa Xaa Leu Phe Ser Xaa Ser Xaa                110                 115                 120Ser Val Asp Xaa Thr Thr Xaa Xaa Gln Gln Leu Xaa Glu Ile Thr                125                 130                 135Xaa Ser Ser Thr Arg Glu Xaa His Val Leu Trp Tyr Ile Ser Leu                140                 145                 150Pro Gly Ala Ala Thr Leu Xaa Ser Met Leu Xaa Xaa Xaa Xaa Xaa                155                 160                 165Xaa Asp Xaa Xaa Xaa Pro Asn Met Xaa Ala Met Xaa Leu Phe Xaa                170                 175                 180Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa Ala Ala Val Gly Gly Arg                185                 190                 195Leu Xaa Xaa Xaa Xaa Ala Ser Lys Xaa Leu Xaa Met Xaa Ser Ser                200                 205                 210Xaa Ser Leu Ser Thr Thr Xaa Xaa Xaa Ser Xaa Xaa Ala Xaa Xaa                215                 220                 225Gly Glu Ile Xaa Ile Xaa His Gly Ser Xaa Met Glu Lys Gln Val                230                 235                 240Asn Ser Phe Xaa Xaa Xaa Ser Thr Ile Arg Xaa Thr Ala Thr Gly                245                 250                 255Gly Lys Pro Gly Xaa Thr Xaa Arg Ile Leu His Gly Pro Asp Ser                260                 265                 270Xaa Xaa Ala Phe Ser Xaa Trp Ala Xaa Ser Leu Leu Xaa Tyr Ala                275                 280                 285Thr Leu Met Asp Phe Xaa Thr Xaa Ser Leu Xaa Xaa Ile Xaa Ala                290                 295                 300Leu Xaa Asp Xaa Pro Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Ala Xaa                305                 310                 315Pro Xaa Xaa Met Xaa Xaa Ser Gln Xaa Ser Ile Pro Xaa Val Asp                320                 325                 330Xaa Val Leu Leu Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly                335                 340                 345Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa                350                 355                 360Xaa Ser Thr Ser Xaa Xaa Tyr Lys Xaa Xaa Gly Gln Phe Xaa Gln                365                 370                 375Arg Xaa His Xaa Ser Val Ala Asp Gly His Xaa Pro Ile Xaa Xaa                380                 385                 390Asp Leu Phe Asp Xaa Gly Xaa Xaa Xaa Xaa Pro Val Gly Xaa Gln                395                 400                 405Xaa Val Trp Asp Xaa Xaa Xaa Xaa Gly Lys Xaa Xaa Xaa Tyr Xaa                410                 415                 420Cys Trp Arg Xaa Xaa Xaa Xaa Gln Gly Tyr Xaa Xaa Xaa Gly Asp                425                 430                 435Val Xaa Met Leu Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro                440                 445                 450Xaa Xaa Xaa Cys Xaa His Xaa Ser Leu Xaa Ala Xaa Xaa Xaa Thr                455                 460                 465Leu Xaa Xaa Xaa Xaa Trp Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa                470                475                 480Xaa Val Ser Leu Trp Xaa Pro Gly Ala Ala Gly Ala Val Ala Ser                485                 490                 495Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser                500                 505                 510Gly Xaa Ile Xaa Xaa Leu Xaa Gly Ser Ile Ala Cys Val Xaa Thr                515                 520                 525Ser Ala Ile Ala Ser Met Xaa Xaa Met Xaa Ser Met Leu Ser Xaa                530                 535His Xaa Gly Met Glu Ala Met Met Ser Lys Leu,wherein Xaa at position 2 is Ala or Arg; Xaa at position 9 is Gin, Lysor Glu; Xaa at position 14 is Pro or Ala; Xaa at position 16 is Val orAsp; Xaa at position 19 is Met or Leu; Xaa at position 22 is Gly or Ser;Xaa at position 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met;Xaa at position 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu;Xaa at position 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is lie orVal; Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys;Xaa at position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; Xaaat position 58 is Thr or Ser; Xaa at position 61 is Arg, Lys or Glu; Xaaat position 73 is Phe or Tyr; Xaa at position 74 is Lys, Glu, Gly, Arg,Met, Leu, His or Asp; Xaa at position 76 is Asp or Gin; Xaa at position79 is Lys or Glu; Xaa at position 80 is Glu or Ser; Xaa at position 82is Glu, lie, Leu, Tyr or Gin; Xaa at position 83 is Glu or Gin; Xaa atposition 84 is Tyr or Phe; Xaa at position 86 is Glu or Gin; Xaa atposition 87 is Lys or Gin; Xaa at position 88 is Met, lie or Leu; Xaa atposition 90 is Gin or Glu; Xaa at position 94 is Val or lie; Xaa atposition 97 is Arg, Asn, Asp, Glu, Gin, Gly or Ser; Xaa at position 98is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie, Met, Phe, Cys,Val or Asn; Xaa at position 103 is Ala or Gly; Xaa at position 105 isLeu or lie; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn,Glu, Cys, Ala or Arg; Xaa at position 112 is Thr or Ser; Xaa at position113 is Asp, Glu or Met; Xaa at position 117 is Thr or Ser; Xaa atposition 121 is Tyr or Phe; Xaa at position 127 is Ala or Thr; Xaa atposition 142 is Arg or Glu; Xaa at position 146 is Arg or Gin; Xaa atposition 147 is Arg, Glu or Gin; Xaa at position 148 is Asp, Phe, Pro,Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gin or Gly; Xaa at position 149is Phe or Val; Xaa at position 150 is Arg, Gin or Glu; Xaa at position151 is Asp, Ser, Ala, Asn, Trp, Val, Gin, Cys, Met, Leu, Arg or Glu; Xaaat position 153 is Leu or lie; Xaa at position 154 is Asn or Asp; Xaa atposition 155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa atposition 162 is Glu, Asp or Gin; Xaa at position 165 is Lys, Glu, Gin,Pro, Thr, Ala, Leu, Gly, Asp, Val, His, lie, Met, Trp, Phe, Tyr or Arg;Xaa at position 166 is Arg or Gin; Xaa at position 167 is Tyr, Trp orCys; Xaa at position 170 is Tyr or His; Xaa at position 171 is Tyr orPhe; Xaa at position 172 is lie, Leu or Val; Xaa at position 173 is Seror Ala; Xaa at position 174 is Glu or Gin; Xaa at position 182 is Asp orGin; Xaa at position 183 is Tyr or Val; Xaa at position 184 is Ser orThr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is Thr, Lysor lie; Xaa at position 191 is Lys or Gin; Xaa at position 193 is Asp orAsn; Xaa at position 196 is Gin, Lys, Asn, Asp, Glu, Ala, lie or Arg;Xaa at position 202 is Ala or Val; Xaa at position 203 is Glu, Thr orHis; Xaa at position 204 is Met or Ala; Xaa at position 206 is Tyr orPhe; Xaa at position 207 is Lys or Gin; Xaa at position 209 is Leu orPro; Xaa at position 210 is Val or lie; Xaa at position 214 is Lys, Seror Gin; Xaa at position 216 is Glu, Gin, Phe, Val, Tyr or Arg; Xaa atposition 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, Gin, Arg, Trp, Met,Ala, Phe, lie, Leu, Cys or Asn; Xaa at position 229 is Arg or Glu; Xaaat position 230 is Ser or Glu; Xaa at position 231 is Asn or Ser; Xaa atposition 236 is Leu or Pro; Xaa at position 245 is Met or Leu; Xaa atposition 247 is Asp or Tyr; Xaa at position 256 is Gin, Lys or Glu; Xaaat position 257 is Gin, lie, Glu, Cys, Ser, His, Trp or Met; Xaa atposition 261 is Gin, Glu or Lys; Xaa at position 264 is Glu or Gin; Xaaat position 268 is Asp or Asn; Xaa at position 276 is Ser or Ala; Xaa atposition 278 is Glu, Asn or Gin; Xaa at position 281 is Gin, Lys or Glu;Xaa at position 282 is Pro or Gly; Xaa at position 284 is Trp or Arg;Xaa at position 287 is Ala or Cys; Xaa at position 289 is Lys, Leu, Val,Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn,Cys, Gly or Ala; Xaa at position 291 is Glu or Gin; Xaa at position 292is Arg or Gin; Xaa at position 293 is Arg, Glu or Gin; Xaa at position294 is Val or Ala; Xaa at position 296 is Leu or lie; Xaa at position297 is Glu or Gin; Xaa at position 298 is Asp or Gin; Xaa at position300 is Phe or Tyr; Xaa at position 302 is Glu or Gin; Xaa at position303 is Phe or Tyr; Xaa at position 305 is Lys or Gin; Xaa at position306 is Gin or Lys; Xaa at position 309 is Gin, Lys or Glu; Xaa atposition 313 is Lys, Gin or Arg; Xaa at position 316 is Lys or Gin; Xaaat position 328 is Lys, Glu or Gin; Xaa at position 331 is Glu, Asn orGin; Xaa at position 333 is Ser, Arg, Gly, Lys, Val, Asn, Ala, His, Gin,Thr, Asp, lie, Leu, Cys or Glu; Xaa at position 334 is Gly, Arg, Lys,lie or Trp; Xaa at position 335 is Ser or Ala; Xaa at position 336 isGly or Ala; Xaa at position 337 is Ala, Val or Gly; Xaa at position 338is Ser, His, Val, Lys, Ala, Gly, Thr, lie, Glu, Met, Arg, Pro, Asp, Asnor Leu; Xaa at position 339 is Glu, Asn, Gin, lie, Pro, Met, Ser, Ala,Cys, Phe, Val, Leu, Asp, Trp, His or Arg; Xaa at position 341 is Leu orVal; Xaa at position 342 is Ala, Ser or Val; Xaa at position 343 is Valor lie; Xaa at position 344 is Phe or Trp; Xaa at position 345 is Asn orHis; Xaa at position 346 is Pro or Ala; Xaa at position 350 is Asn orSer; Xaa at position 351 is Gly or Val; Xaa at position 354 is Met orLeu; Xaa at position 355 is Val, lie or Leu; Xaa at position 359 is Glyor Ala; Xaa at position 362 is Asn or Ser; Xaa at position 364 is Ala orSer; Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is Pheor lie; Xaa at position 375 is Lys or Arg; Xaa at position 380 is Leu orGly; Xaa at position 382 is Val, Asp or Leu; Xaa at position 383 is Leu,lie or Val; Xaa at position 384 is Lys, Ala or Gly; Xaa at position 385is Ala or Gly; Xaa at position 389 is Trp or Tyr; Xaa at position 391 isArg, Leu, Glu, Gin or Asp; Xaa at position 395 is Asp or Cys; Xaa atposition 396 is Ala, Leu, Lys, Asn, Gly, lie, Met, Arg, Tyr, Gin or His;Xaa at position 397 is Gly, Arg or Ala; Xaa at position 398 is Ser, Ginor Cys; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val, Arg, lie,Asn, Phe, Thr, Ala, Asp, Met, Gin or Glu; Xaa at position 402 is Lys,Phe, His, Arg, Trp, Gly, Asn, Leu, Tyr, Thr, Val, Met, Pro or Ala; Xaaat position 403 is Asp, Tyr, Trp, Phe or Glu; Xaa at position 405 is Alaor Ser; Xaa at position 409 is Ala or Pro; Xaa at position 410 is lie orVal; Xaa at position 411 is Pro or Ala; Xaa at position 412 is Pro orAla; Xaa at position 416 is Arg, Glu or Gin; Xaa at position 417 is Ala,Ser or Cys; Xaa at position 418 is Leu or Met; Xaa at position 422 isMet or Val; Xaa at position 426 is Thr or Ser; Xaa at position 436 isAsp or Lys; Xaa at position 437 is Tyr or Val; Xaa at position 438 isVal or Arg; Xaa at position 440 is Val or Leu; Xaa at position 442 isGin, Lys or Glu; Xaa at position 445 is Cys, Leu or Thr; Xaa at position447 is Asp, Lys, Tyr, Ser, Glu, Ile, Gly, Pro, Leu, Phe, Trp or Thr; Xaaat position 448 is Val or Ala; Xaa at position 449 is Gin or Glu; Xaa atposition 452 is Gin, Lys or Glu; Xaa at position 453 is Asn or Asp; Xaaat position 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, Trp, Gin,Gly, His, Asp, Leu, Thr, Pro or Asn; Xaa at position 455 is Val or Ile;Xaa at position 457 is Trp or Asn; Xaa at position 459 is Lys, Met, Val,Trp, Gin, Ile, Thr, Ser, His, Cys, Tyr, Pro, Asn, Ala, Arg or Glu; Xaaat position 460 is Gly or Ala; Xaa at position 461 is Thr or Ser; Xaa atposition 462 is Gly or Ala; Xaa at position 463 is Ala, Ser or Gly; Xaaat position 464 is Arg, Gly, His, Gin, Thr or Phe; Xaa at position 465is Lys, Asn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu;Xaa at position 466 is Asp or Arg; Xaa at position 471 is Gin, Lys orGlu; Xaa at position 497 is Asp or Gin; Xaa at position 499 is Glu orGin; Xaa at position 500 is Arg, Gin or Lys; Xaa at position 502 is Arg,Glu or Gin; Xaa at position 509 is Lys, Gin or Glu; Xaa at position 517is Gin, Cys, Asn, Val or Pro; Xaa at position 518 is Glu or Gin; Xaa atposition 520 is Lys, Gin or Glu; Xaa at position 525 is Gin or Lys; andXaa at position 527 is Gin, Lys, Pro, Cys, Glu, Ser, His, Phe or Trp;wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 3 isan amino acid different from the corresponding amino acid of SEQ ID NO:35 and wherein the PHI-4 polypeptide has increased insecticidal activitycompared to SEQ ID NO: 35.

Embodiment 98 is a polynucleotide encoding a PHI-4 polypeptide, whereinthe PHI-4 polypeptide comprises an amino acid sequence of the formula,

(SEQ ID NO: 4)                5                   10                  15Met Xaa Ser Ala Ala Asn Ala Gly Gln Leu Gly Asn Leu Pro Gly                20                  25                  30Val Thr Ser Met Gly Met Gly Tyr Xaa Val Asn Gly Leu Tyr Ala                35                  40                  45Ser Pro Glu Ser Leu Leu Gly Gln Pro Leu Phe Xaa Xaa Gly Gly                50                  55                  60Xaa Leu Asp Ser Ile Glu Ile Glu Gly Arg Ser Tyr Thr Phe Pro                65                  70                  75Arg Ser Met His Val His Thr Tyr Phe His Ser Asp Phe Xaa Gln                80                  85                  90Asp Val Ser Xaa Glu Ile Xaa Glu Tyr Arg Glu Lys Met Ser Gln                95                  100                 105His Val Gly Val Ser Gly Xaa Xaa Xaa Leu Phe Ser Ala Ser Leu                110                 115                 120Ser Val Asp Xaa Thr Thr Thr Asp Gln Gln Leu Thr Glu Ile Thr                125                 130                 135Tyr Ser Ser Thr Arg Glu Ala His Val Leu Trp Tyr Ile Ser Leu                140                 145                 150Pro Gly Ala Ala Thr Leu Arg Ser Met Leu Arg Xaa Xaa Phe Xaa                155                 160                 165Xaa Asp Xaa Asn Asn Pro Asn Met Pro Ala Met Xaa Leu Phe Xaa                170                 175                 180Xaa Tyr Gly Pro Tyr Xaa Ile Ser Xaa Ala Ala Val Gly Gly Arg                185                 190                 195Leu Xaa Tyr Ser Ala Ala Ser Lys Thr Leu Lys Met Asp Ser Ser                200                 205                 210Xaa Ser Leu Ser Thr Thr Ala Xaa Met Ser Xaa Lys Ala Leu Val                215                 220                 225Gly Glu Ile Lys Ile Xaa His Gly Ser Xaa Met Glu Lys Gln Val                230                 235                 240Asn Ser Phe Arg Ser Asn Ser Thr Ile Arg Leu Thr Ala Thr Gly                245                 250                 255Gly Lys Pro Gly Met Thr Xaa Arg Ile Leu His Gly Pro Asp Ser                260                 265                 270Xaa Xaa Ala Phe Ser Xaa Trp Ala Glu Ser Leu Leu Asp Tyr Ala                275                 280                 285Thr Leu Met Asp Phe Ser Thr Xaa Ser Leu Xaa Pro Ile Trp Ala                290                 295                 300Leu Ala Asp Xaa Pro Glu Arg Xaa Val Glu Leu Glu Asp Ala Phe                305                 310                 315Pro Glu Phe Met Lys Gln Ser Gln Gln Ser Ile Pro Xaa Val Asp                320                 325                 330Lys Val Leu Leu Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly                335                 340                 345Glu Asp Xaa Xaa Ser Xaa Ala Xaa Xaa Asp Leu Ala Xaa Phe Asn                350                 355                 360Xaa Ser Thr Ser Asn Gly Tyr Lys Met Xaa Gly Gln Phe Xaa Gln                365                 370                 375Arg Asn His Ala Ser Val Ala Asp Gly His Ala Pro Ile Phe Lys                380                 385                 390Asp Leu Phe Asp Leu Gly Val Leu Lys Ala Pro Val Gly Trp Gln                395                 400                 405Xaa Val Trp Asp Asp Xaa Gly Ser Gly Lys Xaa Xaa Xaa Tyr Ala                410                 415                 420Cys Trp Arg Ala Ile Xaa Xaa Gln Gly Tyr Xaa Xaa Xaa Gly Asp                425                 430                 435Val Met Met Leu Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Leu Pro                440                 445                 450Asp Tyr Val Cys Xaa His Gln Ser Leu Cys Ala Xaa Val Gln Thr                455                 460                 465Leu Xaa Asn Xaa Xaa Trp Trp Asp Xaa Gly Xaa Xaa Xaa Xaa Xaa                470                 475                 480Asp Val Ser Leu Trp Xaa Pro Gly Ala Ala Gly Ala Val Ala Ser                485                 490                 495Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser                500                 505                 510Gly Asp Ile Glu Xaa Leu Arg Gly Ser Ile Ala Cys Val Xaa Thr                515                 520                 525Ser Ala Ile Ala Ser Met Gln Glu Met Xaa Ser Met Leu Ser Gln                530                 535His Xaa Gly Met Glu Ala Met Met Ser Lys Leu,wherein Xaa at position 2 is Ala or Arg; Xaa at position 24 is Asp orAsn; Xaa at position 42 is Asp or Asn; Xaa at position 43 is Phe or Glu;Xaa at position 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly;Xaa at position 79 is Lys or Glu; Xaa at position 82 is Glu, lie, Leu orTyr; Xaa at position 97 is Arg, Asn, Asp, Glu, Gin or Gly; Xaa atposition 98 is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, lie orMet; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa atposition 147 is Arg or Glu; Xaa at position 148 is Asp, Phe or Pro; Xaaat position 150 is Arg or Gin; Xaa at position 151 is Asp, Ser, Ala orAsn; Xaa at position 153 is Leu or lie; Xaa at position 162 is Glu orGin; Xaa at position 165 is Lys, Glu or Gin; Xaa at position 166 is Argor Gin; Xaa at position 171 is Tyr or Phe; Xaa at position 174 is Glu orGin; Xaa at position 182 is Asp or Gin; Xaa at position 196 is Gin, Lys,Asn or Asp; Xaa at position 203 is Glu, Thr or His; Xaa at position 206is Tyr or Phe; Xaa at position 216 is Glu or Gin; Xaa at position 220 isGlu, His, Asp, Thr, Tyr, Val, Ser or Gin; Xaa at position 247 is Asp orTyr; Xaa at position 256 is Gin or Lys; Xaa at position 257 is Gin orlie; Xaa at position 261 is Gin or Glu; Xaa at position 278 is Glu orAsn; Xaa at position 281 is Gin, Lys or Glu; Xaa at position 289 is Lys,Leu, Val, Pro, Glu, Gin, Tyr, Thr or Asp; Xaa at position 293 is Arg,Glu or Gin; Xaa at position 313 is Lys or Gin; Xaa at position 328 isLys, Glu or Gin; Xaa at position 333 is Ser, Gly, Lys, Val or Asn; Xaaat position 334 is Gly, Arg, Lys or lie; Xaa at position 336 is Gly orAla; Xaa at position 338 is Ser, His, Val, Lys or Ala; Xaa at position339 is Glu, Asn, lie or Pro; Xaa at position 343 is Val or Ile; Xaa atposition 346 is Pro or Ala; Xaa at position 355 is Val or Ile; Xaa atposition 359 is Gly or Ala; Xaa at position 391 is Arg, Glu or Gin; Xaaat position 396 is Ala, Leu, Lys, Asn or Gly; Xaa at position 401 isSer, His, Pro, Gly, Lys, Val or Arg; Xaa at position 402 is Lys, Phe,His, Arg, Gly, Trp, Thr, Asn, Tyr or Met; Xaa at position 403 is Asp orTyr; Xaa at position 411 is Pro or Ala; Xaa at position 412 is Pro orAla; Xaa at position 416 is Arg or Glu; Xaa at position 417 is Ala orSer; Xaa at position 418 is Leu or Met; Xaa at position 426 is Thr orSer; Xaa at position 440 is Val or Leu; Xaa at position 447 is Asp, Lys,Tyr, Ser, Glu or Ile; Xaa at position 452 is Gin, Lys or Glu; Xaa atposition 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, Trp or Gin; Xaaat position 455 is Val or Ile; Xaa at position 459 is Lys, Met, Val,Trp, Gin, Ile or Tyr; Xaa at position 461 is Thr or Ser; Xaa at position462 is Gly or Ala; Xaa at position 463 is Ala or Ser; Xaa at position464 is Arg, Gly or His; Xaa at position 465 is Lys, Asn, Val, Met, Pro,Gly or Arg; Xaa at position 471 is Gin, Lys or Glu; Xaa at position 500is Arg or Gin; Xaa at position 509 is Lys or Gin; Xaa at position 520 isLys, Gin or Glu; and Xaa at position 527 is Gin, Lys, Pro, Cys or Glu;wherein one or more amino acid(s) designated by Xaa in SEQ ID NO: 4 isan amino acid different from the corresponding amino acid of SEQ ID NO:35 and wherein the PHI-4 polypeptide has increased insecticidal activitycompared to SEQ ID NO: 35.

Embodiment 99 is the polynucleotide of embodiments 97 or 98, wherein thePHI-4 polypeptide further comprises one or more amino acid substitutionsat position 86, 359, 464, 465, 466, 467, 468, 499 or 517 of SEQ ID NO: 3or SEQ ID NO: 4.

Embodiment 100 is the polynucleotide of embodiment 99, wherein the aminoacid at position 86 is Glu or Thr; the amino acid at position 359 is Glyor Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp or Asn; theamino acid at position 465 is Lys or Met, the amino acid at position 467is Val, Ala, Leu or Thr; the amino acid at position 468 is Ser or Leu;the amino acid at position 499 is Glu or Ala, or the amino acid atposition 517 is Glu or Arg.

Embodiment 101 is the polynucleotide of any one of embodiments 97-100,wherein the PHI-4 polypeptide further comprises one or more moreconservative amino acid substitution, insertion of one or more aminoacids, deletion of one or more amino acids, and combinations thereof.

Embodiment 102 is the polynucleotide of any one of embodiments 97-101,wherein the insecticidal activity of the PHI-4 polypeptide is increasedabout 1.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 103 is the polynucleotide of any one of embodiments 97-101,wherein the insecticidal activity of the PHI-4 polypeptide is increasedabout 2 fold or greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 104 is the polynucleotide of any one of embodiments 97-101,wherein the insecticidal activity of the PHI-4 polypeptide is increasedabout 2.5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 105 is the polynucleotide of any one of embodiments 97-101,wherein the insecticidal activity of the PHI-4 polypeptide is increasedabout 3 fold or greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 106 is the polynucleotide of any one of embodiments 97-101,wherein the insecticidal activity of the PHI-4 polypeptide is increasedabout 5 fold or greater compared to AXMI-205 (SEQ ID NO: 35).

Embodiment 107 is the polynucleotide of any one of embodiments 97-106,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is against Western Corn Root Worm (WCRW) larvae.

Embodiment 108 is the polynucleotide of any one of embodiments 97-107,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is quantified as a Mean FAE Index.

Embodiment 109 is the polynucleotide of any one of embodiments 97-107,wherein the improved insecticidal activity compared to AXMI-205 (SEQ IDNO: 35) is quantified as an EC50 value.

Embodiment 110 is the polynucleotide of any one of embodiments 97-107,wherein the improved activity compared to AXMI-205 (SEQ ID NO: 35) isquantified as a Mean Deviation Score.

Embodiment 111 is the polynucleotide of any one of embodiments 97-110,wherein the PHI-4 polypeptide has 1 to 54 amino acid substitutions at aposition(s) designated as Xaa in SEQ ID NO: 3 or 4.

Embodiment 112 is the polynucleotide of any one of embodiments 97-110,wherein the PHI-4 polypeptide has 1 to 27 amino acid substitutions at aposition(s) designated as Xaa in SEQ ID NO: 3 or 4.

Embodiment 113 is the polynucleotide of any one of embodiments 97-110,wherein the PHI-4 polypeptide has 1 to 20 amino acid substitutions at aposition(s) designated as Xaa in SEQ ID NO: 3 or 4.

Embodiment 114 is the polynucleotide of any one of embodiments 97-110,wherein the PHI-4 polypeptide has 1 to 15 amino acid substitutions at aposition(s) designated as Xaa in SEQ ID NO: 3 or 4.

Embodiment 115 is the polynucleotide of any one of embodiments 97-114,wherein 1-25 amino acids are deleted from the N-terminus of the PHI-4polypeptide and/or C-terminus of the PHI-4 polypeptide.

Embodiment 116 is the polynucleotide of any one of embodiments 97-114,wherein 1-20 amino acids are deleted from the C-terminus of the PHI-4polypeptide.

Embodiment 117 is a composition, comprising an insecticidally-effectiveamount of the PHI-4 polypeptide of any one of embodiments 1-58.

Embodiment 118 is a method of inhibiting growth or killing an insectpest, comprising contacting the insect pest with the composition ofembodiment 117.

Embodiment 119 is a method for controlling an insect pest populationresistant to a pesticidal protein, comprising contacting the resistantinsect pest population with the composition of embodiment 117.

Embodiment 120 is the method of controlling an insect pest populationresistant to an pesticidal protein of embodiment 119, wherein thepesticidal protein is selected from Cry1Ac, Cry1Ab, Cry1A.105, Cry1Ac,Cry1F, Cry1Fa2, Cry1F, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1,Cry35Ab1, Vip3A, Cry9c, eCry3.1Ab and CBI-Bt.

Embodiment 121 is a transgenic plant or progeny thereof, comprising thepolynucleotide of any one of embodiments 59-116. Embodiment 122 is thetransgenic plant or progeny thereof of embodiment 121, wherein thetransgenic plant is a monocotyledon.

Embodiment 123 is the transgenic plant or progeny thereof of embodiment122, wherein the plant is selected from barley, corn, oat, rice, rye,sorghum, turf grass, sugarcane, wheat, alfalfa, banana, broccoli, bean,cabbage, canola, carrot, cassava, cauliflower, celery, citrus, cotton, acucurbit, eucalyptus, flax, garlic, grape, onion, lettuce, pea, peanut,pepper, potato, poplar, pine, sunflower, safflower, soybean, strawberry,sugar beet, sweet potato, tobacco, tomato ornamental, shrub, nut,chickpea, pigeon pea, millets, hops and pasture grasses.

Embodiment 124 is the transgenic plant or progeny thereof of embodiment123, further comprising one or more additional transgenic traits.

Embodiment 125 is the transgenic plant of embodiment 124, wherein theone or more additional transgenic trait is selected from insectresistance, herbicide resistance, fungal resistance, viral resistance,stress tolerance, disease resistance, male sterility, stalk strength,increased yield, modified starches, improved oil profile, balanced aminoacids, high lysine or methionine, increased digestibility, improvedfiber quality, flowering, ear and seed development, enhancement ofnitrogen utilization efficiency, altered nitrogen responsiveness,drought resistance or tolerance, cold resistance or tolerance, saltresistance or tolerance and increased yield under stress.

Embodiment 126 is seed, grain or processed product thereof of thetransgenic plant of any one of embodiments 121-126, wherein the seed,grain, or processed product thereof comprises the polynucleotide of anyone of embodiments 121-125.

Embodiment 127 is an expression cassette, comprising the polynucleotideof any one of embodiments 59-116 operably linked to one or moreregulatory sequences directing expression of the PHI-4 polypeptide.

Embodiment 128 is a transgenic plant or plant cell, comprising theexpression cassette of embodiment 127.

Embodiment 129 is seed, grain or processed product thereof of thetransgenic plant of embodiment 128, wherein the seed, grain, orprocessed product thereof comprises the recombinant nucleic acidmolecule of embodiment of 1 and the recombinant nucleic acid molecule of24.

Embodiment 130 is the seed of embodiment 129, wherein one or more seedtreatment has been applied to the seed.

Embodiment 131 is a method for expressing in a plant a polynucleotideencoding an insecticidal protein, comprising

(a) inserting into a plant cell the polynucleotide of any one ofembodiment 59-116 encoding the PHI-4 polypeptide;

(b) obtaining a transformed plant cell comprising the nucleic acidsequence of step (a); and

(c) generating from the transformed plant cell a plant capable ofexpressing the polynucleotide encoding the PHI-4 polypeptide.

Embodiment 132 is a method for protecting a plant from an insect pest,comprising expressing in the plant or cell thereof, aninsecticidally-effective amount of the PHI-4 polypeptide of any one ofembodiments 1-58.

Embodiment 133 is a method for controlling an insect pest population,comprising contacting the insect pest population with aninsecticidally-effective amount of the PHI-4 polypeptide of any one ofembodiments 1-58.

Embodiment 134 is a method of inhibiting growth or killing an insectpest, comprising contacting the insect pest with a compositioncomprising an insecticidally-effective amount of the the PHI-4polypeptide of any one of embodiments 1-58.

Embodiment 135 is a method for controlling an insect pest populationresistant to a pesticidal protein, comprising contacting the insect pestpopulation with an insecticidally-effective amount of the PHI-4polypeptide of any one of embodiments 1-58.

Embodiment 136 is a fusion protein comprising the PHI-4 polypeptide ofany one of embodiments 1-58.

Embodiment 137 is a fusion protein comprising the PHI-4 polypeptide ofany one of embodiments 1-58 and a maltose binding protein.

Embodiment 138 is the fusion protein of embodiment 137, wherein themaltose binding protein has an amino acid sequence of SEQ ID NO: 830 orSEQ ID NO: 831.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows FAE analysis of MPB::PHI-4-SFR12-004 (SEQ ID NO 31). Thereference protein is MBP::PHI-4 polypeptide of (SEQ ID NO: 6). The %Response is given on the y axis. The dose of the toxin fragment is givenon the x axis in parts per million (ppm). Half dose (+) and full dose(*) indicate a mean response from six replicate wells at the indicatedconcentration. All data are from a single experiment.

FIG. 2 shows EC50 analysis of MBP::PHI-4 polypeptide of SEQ ID NO 6 andMPB::PHI-4-SFR12-004 (SEQ ID NO 35). The % Response is given on the yaxis. The dose of the toxin fragment is given on the x axis in parts permillion (ppm). The % Response is given on the y axis. The dose of thetoxin fragment is given on the x axis in parts per million (ppm). Theprotein concentration (toxin portion of the protein only) is given onthe x axis. Each symbol indicates a mean response from twenty-fourreplicate wells at the indicated concentration. All data are from asingle experiment. triangles: MBP::PHI-4 (SEQ ID NO: 6); circles:MBP::PHI-4-SFR12-004 (SEQ ID NO: 31).

FIG. 3 shows the amino acids sequence of the C-terminal portion of thePHI-4 polypeptide (SEQ ID NO: 2) is given. Three stretches of sequencecorresponding to nine amino acid motifs that align to the putative sugarbinding loop motif D-X-G-(S/T)-G-X₃-D (SEQ ID NO:40) are highlighted ingrey.

FIG. 4 shows EC50 data for SEQ ID NO: 610, SEQ ID NO: 595, SEQ ID NO:584, SEQ ID NO: 591, SEQ ID NO: 576, SEQ ID NO: 73, SEQ ID NO: 74, SEQID NO: 75, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 150, SEQ ID NO: 150,SEQ ID NO: 149, SEQ ID NO: 167, SEQ ID NO: 167, SEQ ID NO: 164, SEQ IDNO: 164, SEQ ID NO: 170, SEQ ID NO: 170, SEQ ID NO: 795, SEQ ID NO: 794,SEQ ID NO: 784, SEQ ID NO: 799, SEQ ID NO: 785, SEQ ID NO: 788, SEQ IDNO: 786, SEQ ID NO: 796, SEQ ID NO: 787. The x-axis corresponds to themean fold improvement in EC50, relative to MBP::PHI-4 (SEQ ID NO_: 6)from at least three independent EC50 measurements. The y-axiscorresponds to the mean fold improvement of the mean FAE Index from atleast three independent measurements.

FIG. 5. T0 seedlings in the V3-V4 growth stage were challenged asdescribed (Oleson J. et al J. Economic Entomology 98:1-8; 2005) and rootnodal injury scores were recorded. The groups indicated along the x axiswere either controls or were transformed with a plant vector of Example18: 1, untransformed control; 2, positive control transgenic expressingpositive control protein; 3, PHI-4 polypeptide (SEQ ID NO: 22); 4,PHI-4-B09 polypeptide (SEQ ID NO: 23); 5, PHI-4-D09 polypeptide (SEQ IDNO: 24); 6 PHI-4-H08 polypeptide (SEQ ID NO: 25).

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, genera, and reagentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

The present disclosure is drawn to compositions and methods forcontrolling pests. The methods involve transforming organisms with anucleic acid sequence encoding a PHI-4 polypeptide. In particular, thenucleic acid sequences of the embodiments are useful for preparingplants and microorganisms possessing pesticidal activity. Thus,transformed bacteria, plants, plant cells, plant tissues and seeds areprovided. Compositions are pesticidal nucleic acids and proteins ofbacterial species. The nucleic acid sequences find use in theconstruction of expression vectors for subsequent transformation intoorganisms of interest, as probes for the isolation of other homologous(or partially homologous) genes, and for the generation of altered PHI-4polypeptides by methods known in the art, such as site directedmutagenesis, domain swapping or DNA shuffling. The PHI-4 polypeptidesfind use in controlling, inhibiting growth or killing Lepidopteran,Coleopteran, Dipteran, fungal, Hemipteran, and nematode pest populationsand for producing compositions with pesticidal activity. Insect pests ofinterest include, but are not limited to, the superfamily of stink bugsand other related insects including, but not limited to, speciesbelonging to the family Pentatomidae (Nezara viridula, Halyomorphahalys, Piezodorus guildini, Euschistus servus, Acrosternum hilare,Euschistus heros, Euschistus tristigmus, Acrosternum hilare, Dichelopsfurcatus, Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), thefamily Plataspidae (Megacopta cribraria—Bean plataspid), and the familyCydnidae (Scaptocoris castanea—Root stink bug) and Lepidoptera speciesincluding but not limited to: diamond-back moth, e.g., Helicoverpa zeaBoddie; soybean looper, e.g., Pseudoplusia includens Walker and velvetbean caterpillar e.g., Anticarsia gemmatalis Hubner.

By “pesticidal toxin” or “pesticidal protein” is intended a toxin thathas toxic activity against one or more pests, including, but not limitedto, members of the Lepidoptera, Diptera, Hemiptera and Coleoptera ordersor the Nematoda phylum or a protein that has homology to such a protein.Pesticidal proteins have been isolated from organisms including, forexample, Bacillus sp., Pseudomonas sp., Photorhabdus sp., Xenorhabdussp., Clostridium bifermentans and Paenibacillus popilliae. Pesticidalproteins include but are not limited to: insecticidal proteins fromPseudomonas sp. such as PSEEN3174 (Monalysin, (2011) PLoS Pathogens,7:1-13) from Pseudomonas protegens strain CHAO and Pf-5 (previouslyfluorescens) (Pechy-Tarr, (2008) Environmental Microbiology10:2368-2386: GenBank Accession No. EU400157); from PseudomonasTaiwanensis (Liu, et al., (2010) J. Agric. Food Chem. 58:12343-12349)and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals ofMicrobiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. OrganCult. 89:159-168); insecticidal proteins from Photorhabdus sp. andXenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxinology Journal3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro.67:2062-2069), U.S. Pat. Nos. 6,048,838, and 6,379,946; and δ-endotoxinsincluding, but not limited to, the cry1, cry2, cry3, cry4, cry5, cry6,cry7, cry8, cry9, cry10, cry11, cry12, cry13, cry14, cry15, cry16,cry17, cry18, cry19, cry20, cry21, cry22, cry23, cry24, cry25, cry26,cry27, cry 28, cry 29, cry 30, cry31, cry32, cry33, cry34, cry35, cry36,cry37, cry38, cry39, cry40, cry41, cry42, cry43, cry44, cry45, cry 46,cry47, cry49, cry 51 and cry55 classes of δ-endotoxin genes and the B.thuringiensis cytolytic cyt1 and cyt2 genes. Members of these classes ofB. thuringiensis insecticidal proteins include, but are not limited toCry1Aa1 (Accession # M11250), Cry1Aa2 (Accession # M10917), Cry1Aa3(Accession # D00348), Cry1Aa4 (Accession # X13535), Cry1Aa5 (Accession #D17518), Cry1Aa6 (Accession # U43605), Cry1Aa7 (Accession # AF081790),Cry1Aa8 (Accession #126149), Cry1Aa9 (Accession # AB026261), Cry1Aa10(Accession # AF154676), Cry1Aa11 (Accession # Y09663), Cry1Aa12(Accession # AF384211), Cry1Aa13 (Accession # AF510713), Cry1Aa14(Accession # AY197341), Cry1Aa15 (Accession #DQ062690), Cry1Ab1(Accession # M13898), Cry1Ab2 (Accession # M12661), Cry1Ab3 (Accession #M15271), Cry1Ab4 (Accession # D00117), Cry1Ab5 (Accession # X04698),Cry1Ab6 (Accession # M37263), Cry1Ab7 (Accession # X13233), Cry1Ab8(Accession #M16463), Cry1Ab9 (Accession # X54939), Cry1Ab10 (Accession #A29125), Cry1Ab11 (Accession #112419), Cry1Ab12 (Accession # AF059670),Cry1Ab13 (Accession #AF254640), Cry1Ab14 (Accession # U94191), Cry1Ab15(Accession # AF358861), Cry1Ab16 (Accession # AF375608), Cry1Ab17(Accession # AAT46415), Cry1Ab18 (Accession # AAQ88259), Cry1Ab19(Accession # AY847289), Cry1Ab20 (Accession #DQ241675), Cry1Ab21(Accession # EF683163), Cry1Ab22 (Accession # ABW87320), Cry1Ab-like(Accession # AF327924), Cry1Ab-like (Accession # AF327925), Cry1Ab-like(Accession # AF327926), Cry1Ab-like (Accession # DQ781309), Cry1Ac1(Accession #M11068), Cry1Ac2 (Accession # M35524), Cry1Ac3 (Accession #X54159), Cry1Ac4 (Accession # M73249), Cry1Ac5 (Accession # M73248),Cry1Ac6 (Accession # U43606), Cry1Ac7 (Accession # U87793), Cry1Ac8(Accession # U87397), Cry1Ac9 (Accession #U89872), Cry1Ac10 (Accession #AJ002514), Cry1Ac11 (Accession # AJ130970), Cry1Ac12 (Accession#112418), Cry1Ac13 (Accession # AF148644), Cry1Ac14 (Accession #AF492767), Cry1Ac15 (Accession # AY122057), Cry1Ac16 (Accession#AY730621), Cry1Ac17 (Accession # AY925090), Cry1Ac18 (Accession #DQ023296), Cry1Ac19 (Accession # DQ195217), Cry1Ac20 (Accession #DQ285666), Cry1Ac21 (Accession # DQ062689), Cry1Ac22 (Accession #EU282379), Cry1Ac23 (Accession #AM949588), Cry1Ac24 (Accession #ABL01535), Cry1Ad1 (Accession # M73250), Cry1Ad2 (Accession # A27531),Cry1Ae1 (Accession # M65252), Cry1Af1 (Accession #U82003), Cry1Ag1(Accession # AF081248), Cry1Ah1 (Accession # AF281866), Cry1Ah2(Accession # DQ269474), Cry1Ai1 (Accession # AY174873), Cry1A-like(Accession #AF327927), Cry1 Ba1 (Accession # X06711), Cry1 Ba2(Accession # X95704), Cry1 Ba3 (Accession # AF368257), Cry1 Ba4(Accession # AF363025), Cry1 Ba5 (Accession #ABO20894), Cry1 Ba6(Accession # ABL60921), Cry1 Bb1 (Accession # L32020), Cry1 Bc1(Accession # Z46442), Cry1 Bd1 (Accession # U70726), Cry1 Bd2 (Accession#AY138457), Cry1Be1 (Accession # AF077326), Cry1Be2 (Accession #AAQ52387), Cry1Bf1 (Accession # AX189649), Cry1Bf2 (Accession #AAQ52380), Cry1Bg1 (Accession # AY176063), Cry1Ca1 (Accession # X07518),Cry1Ca2 (Accession #X13620), Cry1Ca3 (Accession # M73251), Cry1Ca4(Accession # A27642), Cry1Ca5 (Accession # X96682), Cry1Ca6 [1](Accession # AF215647), Cry1Ca7 (Accession #AY015492), Cry1Ca8(Accession # AF362020), Cry1Ca9 (Accession # AY078160), Cry1Ca10(Accession # AF540014), Cry1Ca11 (Accession # AY955268), Cry1Cb1(Accession # M97880), Cry1Cb2 (Accession # AY007686), Cry1Cb3 (Accession#EU679502), Cry1Cb-like (Accession # AAX63901), Cry1Da1 (Accession #X54160), Cry1Da2 (Accession #176415), Cry1 Db1 (Accession # Z22511),Cry1 Db2 (Accession #AF358862), Cry1 Dc1 (Accession # EF059913), Cry1Ea1 (Accession # X53985), Cry1 Ea2 (Accession # X56144), Cry1 Ea3(Accession # M73252), Cry1 Ea4 (Accession # U94323), Cry1Ea5 (Accession# A15535), Cry1Ea6 (Accession # AF202531), Cry1Ea7 (Accession #AAW72936), Cry1 Ea8 (Accession # ABX11258), Cry1 Eb1 (Accession #M73253), Cry1 Fa1 (Accession # M63897), Cry1 Fa2 (Accession # M73254),Cry1 Fb1 (Accession #Z22512), Cry1 Fb2 (Accession # AB012288), Cry1 Fb3(Accession # AF062350), Cry1 Fb4 (Accession #173895), Cry1 Fb5(Accession # AF336114), Cry1 Fb6 (Accession #EU679500), Cry1Fb7(Accession # EU679501), Cry1Ga1 (Accession # Z22510), Cry1Ga2 (Accession# Y09326), Cry1Gb1 (Accession # U70725), Cry1Gb2 (Accession #AF288683),Cry1Gc (Accession # AAQ52381), Cry1Ha1 (Accession # Z22513), Cry1 Hb1(Accession # U35780), Cry1H-like (Accession # AF182196), Cry1Ia1(Accession #X62821), Cry1Ia2 (Accession # M98544), Cry1Ia3 (Accession #L36338), Cry1Ia4 (Accession # L49391), Cry1 Ia5 (Accession # Y08920),Cry1 Ia6 (Accession # AF076953), Cry1 Ia7 (Accession # AF278797), Cry1Ia8 (Accession # AF373207), Cry1 Ia9 (Accession # AF521013), Cry1Ia10(Accession # AY262167), Cry1Ia11 (Accession # AJ315121), Cry1Ia12(Accession # AAV53390), Cry1Ia13 (Accession # ABF83202), Cry1Ia14(Accession # EU887515), Cry1Ib1 (Accession # U07642), Cry1Ib2 (Accession#ABW88019), Cry1Ib3 (Accession # EU677422), Cry1Ic1 (Accession #AF056933), Cry1Ic2 (Accession # AAE71691), Cry1Id1 (Accession #AF047579), Cry1Ie1 (Accession # AF211190), Cry1If1 (Accession #AAQ52382), Cry1I-like (Accession #190732), Cry1I-like (Accession #DQ781310), Cry1Ja1 (Accession # L32019), Cry1Jb1 (Accession #U31527),Cry1Jc1 (Accession #190730), Cry1Jc2 (Accession # AAQ52372), Cry1Jd1(Accession # AX189651), Cry1Ka1 (Accession # U28801), Cry1La1 (Accession#AAS60191), Cry1-like (Accession #190729), Cry2Aa1 (Accession # M31738),Cry2Aa2 (Accession # M23723), Cry2Aa3 (Accession # D86064), Cry2Aa4(Accession #AF047038), Cry2Aa5 (Accession # AJ132464), Cry2Aa6(Accession # AJ132465), Cry2Aa7 (Accession # AJ132463), Cry2Aa8(Accession # AF252262), Cry2Aa9 (Accession # AF273218), Cry2Aa10(Accession # AF433645), Cry2Aa11 (Accession #AAQ52384), Cry2Aa12(Accession # DQ977646), Cry2Aa13 (Accession # ABL01536), Cry2Aa14(Accession # ACF04939), Cry2Ab1 (Accession # M23724), Cry2Ab2 (Accession# X55416), Cry2Ab3 (Accession # AF164666), Cry2Ab4 (Accession#AF336115), Cry2Ab5 (Accession # AF441855), Cry2Ab6 (Accession #AY297091), Cry2Ab7 (Accession # DQ119823), Cry2Ab8 (Accession #DQ361266), Cry2Ab9 (Accession # DQ341378), Cry2Ab10 (Accession #EF157306), Cry2Ab11 (Accession #AM691748), Cry2Ab12 (Accession #ABM21764), Cry2Ab13 (Accession # EU909454), Cry2Ab14 (Accession #EU909455), Cry2Ac1 (Accession # X57252), Cry2Ac2 (Accession # AY007687),Cry2Ac3 (Accession # AAQ52385), Cry2Ac4 (Accession # DQ361267), Cry2Ac5(Accession # DQ341379), Cry2Ac6 (Accession # DQ359137), Cry2Ac7(Accession # AM292031), Cry2Ac8 (Accession # AM421903), Cry2Ac9(Accession #AM421904), Cry2Ac10 (Accession # BI 877475), Cry2Ac11(Accession # AM689531), Cry2Ac12 (Accession # AM689532), Cry2Ad11(Accession # AF200816), Cry2Ad2 (Accession # DQ358053), Cry2Ad3(Accession # AM268418), Cry2Ad4 (Accession #AM490199), Cry2Ad5(Accession # AM765844), Cry2Ae1 (Accession # AAQ52362), Cry2Af1(Accession # EF439818), Cry2Ag (Accession # ACH91610), Cry2Ah (Accession# EU939453), Cry3Aa1 (Accession # M22472), Cry3Aa2 (Accession # J02978),Cry3Aa3 (Accession # Y00420), Cry3Aa4 (Accession # M30503), Cry3Aa5(Accession # M37207), Cry3Aa6 (Accession # U10985), Cry3Aa7 (Accession #AJ237900), Cry3Aa8 (Accession # AAS79487), Cry3Aa9 (Accession #AAW05659), Cry3Aa10 (Accession # AAU29411), Cry3Aa11 (Accession #AY882576), Cry3Aa12 (Accession # ABY49136), Cry3Ba11 (Accession #X17123), Cry3Ba2 (Accession # A07234), Cry3Bb1 (Accession # M89794),Cry3Bb2 (Accession # U31633), Cry3Bb3 (Accession #115475), Cry3Ca1(Accession #X59797), Cry4Aa1 (Accession # Y00423), Cry4Aa2 (Accession #D00248), Cry4Aa3 (Accession # AL731825), Cry4A-like (Accession #DQ078744), Cry4Ba1 (Accession #X07423), Cry4Ba2 (Accession # X07082),Cry4Ba3 (Accession # M20242), Cry4Ba4 (Accession # D00247), Cry4Ba5(Accession # AL731825), Cry4Ba-like (Accession #ABC47686), Cry4Ca1(Accession # EU646202), Cry5Aa1 (Accession # L07025), Cry5Ab1 (Accession# L07026), Cry5Ac1 (Accession #134543), Cry5Ad1 (Accession # EF219060),Cry5Ba1 (Accession # U19725), Cry5Ba2 (Accession # EU121522), Cry6Aa1(Accession # L07022), Cry6Aa2 (Accession # AF499736), Cry6Aa3 (Accession# DQ835612), Cry6Ba1 (Accession # L07024), Cry7Aa1 (Accession # M64478),Cry7Ab1 (Accession #U04367), Cry7Ab2 (Accession # U04368), Cry7Ab3(Accession # BI 1015188), Cry7Ab4 (Accession # EU380678), Cry7Ab5(Accession # ABX79555), Cry7Ab6 (Accession #FJ194973), Cry7Ba1(Accession # ABB70817), Cry7Ca1 (Accession # EF486523), Cry8Aa1(Accession # U04364), Cry8Ab1 (Accession # EU044830), Cry8Ba1 (Accession# U04365), Cry8Bb1 (Accession # AX543924), Cry8Bc1 (Accession #AX543926), Cry8Ca1 (Accession # U04366), Cry8Ca2 (Accession # AAR98783),Cry8Ca3 (Accession # EU625349), Cry8Da1 (Accession # AB089299), Cry8Da2(Accession # BD133574), Cry8Da3 (Accession # BD133575), Cry8Db1(Accession # AB303980), Cry8Ea1 (Accession # AY329081), Cry8Ea2(Accession # EU047597), Cry8Fa1 (Accession #AY551093), Cry8Ga1(Accession # AY590188), Cry8Ga2 (Accession # DQ318860), Cry8Ga3(Accession # FJ198072), Cry8Ha1 (Accession # EF465532), Cry8Ia1(Accession # EU381044), Cry8Ja1 (Accession # EU625348), Cry8 like(Accession #ABS53003), Cry9Aa1 (Accession # X58120), Cry9Aa2 (Accession# X58534), Cry9Aa like (Accession # AAQ52376), Cry9Ba1 (Accession #X75019), Cry9Bb1 (Accession #AY758316), Cry9Ca1 (Accession # Z37527),Cry9Ca2 (Accession # AAQ52375), Cry9Da1 (Accession # D85560), Cry9Da2(Accession # AF042733), Cry9Db1 (Accession # AY971349), Cry9Ea1(Accession # AB011496), Cry9Ea2 (Accession # AF358863), Cry9Ea3(Accession # EF157307), Cry9Ea4 (Accession # EU760456), Cry9Ea5(Accession # EU789519), Cry9Ea6 (Accession # EU887516), Cry9Eb1(Accession #AX189653), Cry9Ec1 (Accession # AF093107), Cry9Ed1(Accession # AY973867), Cry9 like (Accession # AF093107), Cry10Aa1(Accession # M12662), Cry10Aa2 (Accession #E00614), Cry10Aa3 (Accession# AL731825), Cry10A like (Accession # DQ167578), Cry11 Aa1 (Accession #M31737), Cry11Aa2 (Accession # M22860), Cry11Aa3 (Accession # AL731825),Cry11Aa-like (Accession # DQ166531), Cry11Ba1 (Accession #X86902),Cry11Bb1 (Accession # AF017416), Cry12Aa1 (Accession # L07027), Cry13Aa1(Accession # L07023), Cry14Aa1 (Accession # U13955), Cry15Aa1 (Accession# M76442), Cry16Aa1 (Accession # X94146), Cry17Aa1 (Accession # X99478),Cry18Aa1 (Accession # X99049), Cry18Ba1 (Accession # AF169250), Cry18Ca1(Accession # AF169251), Cry19Aa1 (Accession # Y07603), Cry19Ba1(Accession #D88381), Cry20Aa1 (Accession # U82518), Cry21Aa1 (Accession#132932), Cry21Aa2 (Accession #166477), Cry21 Ba1 (Accession #AB088406), Cry22Aa1 (Accession #134547), Cry22Aa2 (Accession #AX472772), Cry22Aa3 (Accession # EU715020), Cry22Ab1 (Accession #AAK50456), Cry22Ab2 (Accession # AX472764), Cry22Ba1 (Accession #AX472770), Cry23Aa1 (Accession # AAF76375), Cry24Aa1 (Accession#U88188), Cry24Ba1 (Accession # BAD32657), Cry24Ca1 (Accession #AM158318), Cry25Aa1 (Accession # U88189), Cry26Aa1 (Accession #AF122897), Cry27Aa1 (Accession # AB023293), Cry28Aa1 (Accession #AF132928), Cry28Aa2 (Accession #AF285775), Cry29Aa1 (Accession #AJ251977), Cry30Aa1 (Accession # AJ251978), Cry30Ba1 (Accession #BAD00052), Cry30Ca1 (Accession # BAD67157), Cry30Da1 (Accession #EF095955), Cry30Db1 (Accession # BAE80088), Cry30Ea1 (Accession#EU503140), Cry30Fa1 (Accession # EU751609), Cry30Ga1 (Accession #EU882064), Cry31Aa1 (Accession # AB031065), Cry31Aa2 (Accession #AY081052), Cry31Aa3 (Accession # AB250922), Cry31Aa4 (Accession #AB274826), Cry31Aa5 (Accession #AB274827), Cry31Ab1 (Accession #AB250923), Cry31Ab2 (Accession # AB274825), Cry31Ac1 (Accession #AB276125), Cry32Aa1 (Accession # AY008143), Cry32Ba1 (Accession #BAB78601), Cry32Ca1 (Accession # BAB78602), Cry32Da1 (Accession#BAB78603), Cry33Aa1 (Accession # AAL26871), Cry34Aa1 (Accession #AAG50341), Cry34Aa2 (Accession # AAK64560), Cry34Aa3 (Accession #AY536899), Cry34Aa4 (Accession # AY536897), Cry34Ab1 (Accession #AAG41671), Cry34Ac1 (Accession #AAG50118), Cry34Ac2 (Accession #AAK64562), Cry34Ac3 (Accession # AY536896), Cry34Ba1 (Accession #AAK64565), Cry34Ba2 (Accession # AY536900), Cry34Ba3 (Accession #AY536898), Cry35Aa1 (Accession # AAG50342), Cry35Aa2 (Accession#AAK64561), Cry35Aa3 (Accession # AY536895), Cry35Aa4 (Accession #AY536892), Cry35Ab1 (Accession # AAG41672), Cry35Ab2 (Accession #AAK64563), Cry35Ab3 (Accession # AY536891), Cry35Ac1 (Accession #AAG50117), Cry35Ba1 (Accession #AAK64566), Cry35Ba2 (Accession #AY536894), Cry35Ba3 (Accession # AY536893), Cry36Aa1 (Accession #AAK64558), Cry37Aa1 (Accession # AAF76376), Cry38Aa1 (Accession #AAK64559), Cry39Aa1 (Accession # BAB72016), Cry40Aa1 (Accession#BAB72018), Cry40Ba1 (Accession # BAC77648), Cry40Ca1 (Accession #EU381045), Cry40Da1 (Accession # EU596478), Cry41Aa1 (Accession #AB116649), Cry41Ab1 (Accession # AB116651), Cry42Aa1 (Accession #AB116652), Cry43Aa1 (Accession #AB115422), Cry43Aa2 (Accession #AB176668), Cry43Ba1 (Accession # AB115422), Cry43-like (Accession #AB115422), Cry44Aa (Accession # BAD08532), Cry45Aa (Accession #BAD22577), Cry46Aa (Accession # BAC79010), Cry46Aa2 (Accession#BAG68906), Cry46Ab (Accession # BAD35170), Cry47Aa (Accession #AY950229), Cry48Aa (Accession # AJ841948), Cry48Aa2 (Accession #AM237205), Cry48Aa3 (Accession # AM237206), Cry48Ab (Accession #AM237207), Cry48Ab2 (Accession #AM237208), Cry49Aa (Accession #AJ841948), Cry49Aa2 (Accession # AM237201), Cry49Aa3 (Accession #AM237203), Cry49Aa4 (Accession # AM237204), Cry49Ab1 (Accession #AM237202), Cry50Aa1 (Accession # AB253419), Cry51Aa1 (Accession#DQ836184), Cry52Aa1 (Accession # EF613489), Cry53Aa1 (Accession #EF633476), Cry54Aa1 (Accession # EU339367), Cry55Aa1 (Accession #EU121521), Cry55Aa2 (Accession # AAE33526), Cyt1Aa (GenBank Accession #X03182), Cyt1Ab (GenBank Accession # X98793), Cyt1B (GenBank Accession #U37196), Cyt2A (GenBank Accession # Z14147), Cyt2B (GenBank Accession #U52043).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of cryproteins such as Cry1A, Cry3A) of U.S. Pat. Nos. 8,304,604 and8,304,605, Cry1B of U.S. patent application Ser. No. 10/525,318; Cry1Cof U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960,6,218,188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and6,713,063); a Cry2 protein such as Cry2Ab protein of U.S. Pat. No.7,064,249); a Cry3A protein including but not limited to an engineeredhybrid insecticidal protein (eHIP) created by fusing unique combinationsof variable regions and conserved blocks of at least two different Cryproteins (US Patent Application Publication Number 2010/0017914); a Cry4protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos.7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B,Cry9C, Cry9D, Cry9E, and Cry9F families; a Cry15 protein of Naimov, etal., (2008) Applied and Environmental Microbiology 74:7145-7151; aCry22, a Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and6,340,593; a CryET33 and cryET34 protein of U.S. Pat. Nos. 6,248,535,6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 andCryET34 homologs of US Patent Publication Number 2006/0191034,2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1protein of U.S. Pat. Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or relatedtoxin; TIC807 of US 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812,TIC127, TIC128 of PCT US 2006/033867; AXMI-027, AXMI-036, and AXMI-038of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 ofU.S. Pat. No. 7,923,602; AXMI-018, AXMI-020, and AXMI-021 of WO2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO 2005/021585;AXMI-008 of US 2004/0250311; AXMI-006 of US 2004/0216186; AXMI-007 of US2004/0210965; AXMI-009 of US 2004/0210964; AXMI-014 of US 2004/0197917;AXMI-004 of US 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457;AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 ofUS20110023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019,AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023,AXMI-041, AXMI-063, and AXMI-064 of US 2011/0263488; AXMI-R1 and relatedproteins of US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z andAXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227,AXMI228, AXMI229, AXMI230, and AXMI231 of WO11/103247; AXMI-115,AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US 2010/0298211;AXMI-066 and AXMI-076 of US20090144852; AXMI128, AXMI130, AXMI131,AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148,AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158,AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171,AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179,AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189of U.S. Pat. No. 8,318,900; AXMI0079, AXMI080, AXMI0081, AXMI0082,AXMI091, AXMI0092, AXMI0096, AXMI0097, AXMI0098, AXMI0099, AXMI100,AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110,AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120,AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127,AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137of US 2010/0005543; cry proteins such as Cry1A and Cry3A having modifiedproteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aa andCry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 of USPatent Application Publication Number 2011/0064710. Other Cry proteinsare well known to one skilled in the art (see, Crickmore, et al.,“Bacillus thuringiensis toxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/which can be accessed on theworld-wide web using the “www” prefix). The insecticidal activity of Cryproteins is well known to one skilled in the art (for review, see, vanFrannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cryproteins as transgenic plant traits is well known to one skilled in theart and Cry-transgenic plants including but not limited to Cry1Ac,Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab,Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c andCBI-Bt have received regulatory approval (see, Sanahuja, (2011) PlantBiotech Journal 9:283-300 and the CERA (2010) GM Crop Database Centerfor Environmental Risk Assessment (CERA), ILSI Research Foundation,Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database whichcan be accessed on the world-wide web using the “www” prefix). More thanone pesticidal proteins well known to one skilled in the art can also beexpressed in plants such as Vip3Ab & Cry1 Fa (US2012/0317682), Cry1BE &Cry1F (US2012/0311746), Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa(US2012/0317681), Cry1DA & Cry1BE (US2012/0331590), Cry1DA & Cry1Fa(US2012/0331589), Cry1AB & Cry1 BE (US2012/0324606), and Cry1Fa &Cry2Aa, Cry1 I or Cry1E (US2012/0324605). Pesticidal proteins alsoinclude insecticidal lipases including lipid acyl hydrolases of U.S.Pat. No. 7,491,869, and cholesterol oxidases such as from Streptomyces(Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413).Pesticidal proteins also include VIP (vegetative insecticidal proteins)toxins of U.S. Pat. Nos. 5,877,012, 6,107,279 6,137,033, 7,244,820,7,615,686, and 8,237,020 and the like. Other VIP proteins are well knownto one skilled in the art (see,lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but not limited to lycotoxin-1peptides and mutants thereof (U.S. Pat. No. 8,334,366).

In some embodiments the PHI-4 polypeptides include amino acid sequencesdeduced from the full-length nucleic acid sequences disclosed herein,and amino acid sequences that are shorter than the full-lengthsequences, either due to the use of an alternate downstream start siteor due to processing that produces a shorter protein having pesticidalactivity. Processing may occur in the organism the protein is expressedin, or in the pest after ingestion of the protein.

Thus, provided herein are novel isolated or recombinant nucleic acidsequences that confer pesticidal activity. Also provided are the aminoacid sequences of PHI-4 polypeptides. The protein resulting fromtranslation of these PHI-4 polypeptide genes allows cells to control orkill pests that ingest it.

Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect pertains to isolated or recombinant nucleic acid moleculescomprising nucleic acid sequences encoding PHI-4 polypeptides andpolypeptides or biologically active portions thereof, as well as nucleicacid molecules sufficient for use as hybridization probes to identifynucleic acid molecules encoding proteins with regions of sequencehomology. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA,plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” or “recombinant” nucleic acid molecule (or DNA) is usedherein to refer to a nucleic acid sequence (or DNA) that is no longer inits natural environment, for example in an in vitro or in a recombinantbacterial or plant host cell. In some embodiments, an “isolated” or“recombinant” nucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forpurposes of the disclosure, “isolated” or “recombinant” when used torefer to nucleic acid molecules excludes isolated chromosomes. Forexample, in various embodiments, the recombinant nucleic acid moleculeencoding a PHI-4 polypeptide can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleic acid sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived.

A variety of polynucleotides encoding a PHI-4 polypeptide(s) or relatedproteins are contemplated. Such polynucleotides are useful forproduction of PHI-4 polypeptides in host cells when operably linked tosuitable promoter, transcription termination and/or polyadenylationsequences. Such polynucleotides are also useful as probes for isolatinghomologous or substantially homologous polynucleotides encoding PHI-4polypeptides or related proteins.

The present invention provides isolated or recombinant polynucleotidesthat encode any of the PHI-4 polypeptides disclosed herein. Those havingordinary skill in the art will readily appreciate that due to thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding β-glucosidase polypeptides of the present invention exist.Table 1 is a Codon Table that provides the synonymous codons for eachamino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the invention where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that U in anRNA sequence corresponds to T in a DNA sequence.

TABLE 1 Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Asparticacid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUCUUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine IleI AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

Such “silent variations” are one species of “conservative” variation.One of ordinary skill in the art will recognize that each codon in anucleic acid (except AUG, which is ordinarily the only codon formethionine) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention contemplates and provides each andevery possible variation of nucleic acid sequence encoding a polypeptideof the invention that could be made by selecting combinations based onpossible codon choices. These combinations are made in accordance withthe standard triplet genetic code (set forth in Table 1), as applied tothe polynucleotide sequences of the present invention.

A group of two or more different codons that, when translated in thesame context, all encode the same amino acid, are referred to herein as“synonymous codons.” Polynucleotides encoding PHI-4 polypeptides of thepresent disclosure may be codon optimized for expression in a particularhost organism by modifying the polynucleotides to conform with theoptimum codon usage of the desired host organism. Those having ordinaryskill in the art will recognize that tables and other referencesproviding preference information for a wide range of organisms arereadily available.

Polynucleotides encoding a PHI-4 polypeptide can also be synthesized denovo from a PHI-4 polypeptide sequence. The sequence of thepolynucleotide gene can be deduced from a PHI-4 polypeptide sequencethrough use of the genetic code. Computer programs such as“BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can beused to convert a peptide sequence to the corresponding nucleotidesequence encoding the peptide. Examples of PHI-4 polypeptide sequencesthat can be used to obtain corresponding nucleotide encoding sequencesinclude, but are not limited to, the PHI-4 polypeptide sequence of SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-819. Furthermore,synthetic PHI-4 polynucleotide sequences of the invention can bedesigned so that they will be expressed in plants. U.S. Pat. No.5,500,365 describes a method for synthesizing plant genes to improve theexpression level of the protein encoded by the synthesized gene. Thismethod relates to the modification of the structural gene sequences ofthe exogenous transgene, to cause them to be more efficientlytranscribed, processed, translated and expressed by the plant. Featuresof genes that are expressed well in plants include elimination ofsequences that can cause undesired intron splicing or polyadenylation inthe coding region of a gene transcript while retaining substantially theamino acid sequence of the toxic portion of the insecticidal protein. Asimilar method for obtaining enhanced expression of transgenes inmonocotyledonous plants is disclosed in U.S. Pat. No. 5,689,052.

In some embodiments the nucleic acid molecule encoding a PHI-4polypeptide is a polynucleotide having the sequence set forth in SEQ IDNO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NOS: 24-30 and variants,fragments and complements thereof. By “complement” is intended a nucleicacid sequence that is sufficiently complementary to a given nucleic acidsequence such that it can hybridize to the given nucleic acid sequenceto thereby form a stable duplex. In some embodiments the nucleic acidmolecule encoding a PHI-4 polypeptide is a nucleic acid molecule havingthe sequence set forth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQID NOS: 24-30. The corresponding amino acid sequences for theinsecticidal protein encoded by these nucleic acid sequences are setforth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NOS: 24-30.

In some embodiments the nucleic acid molecule encoding a PHI-4polypeptide is a polynucleotide having a nucleotide sequence encoding apolypeptide comprising an amino acid sequence having at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater sequence identity to the amino acidsequence of SEQ ID NO: 35, wherein the polypeptide has pesticidalactivity. In some embodiments the nucleic acid molecule encoding a PHI-4polypeptide is a polynucleotide having a nucleotide sequence encoding apolypeptide comprising an amino acid sequence having 80% and 95%identity, to the amino acid sequence of SEQ ID NO: 35, wherein thepolypeptide has pesticidal activity.

In some embodiments the nucleic acid molecule encoding a PHI-4polypeptide is a polynucleotide having a nucleotide sequence encoding apolypeptide comprising an amino acid sequence having at least 80%identity, to the amino acid sequence of SEQ ID NO: 51-819, wherein thepolypeptide has pesticidal activity.

In some embodiments the nucleic acid molecule encoding a PHI-4polypeptide is a polynucleotide having a nucleotide sequence encoding apolypeptide comprising an amino acid sequence SEQ ID NO: 4, wherein Xaaat position 9 is Gin, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaaat position 16 is Val or Asp; Xaa at position 19 is Met or Leu; Xaa atposition 22 is Gly or Ser; Xaa at position 24 is Asp, Asn or Gin; Xaa atposition 36 is Leu or Met; Xaa at position 42 is Asp, Asn or Gin; Xaa atposition 43 is Phe or Glu; Xaa at position 46 is Glu, Asp, Asn or Gly;Xaa at position 50 is Ile or Val; Xaa at position 51 is Glu or Gin; Xaaat position 55 is Arg or Lys; Xaa at position 56 is Ser or Thr; Xaa atposition 57 is Tyr or Phe; Xaa at position 58 is Thr or Ser; Xaa atposition 61 is Arg, Lys or Glu; Xaa at position 73 is Phe or Tyr; Xaa atposition 74 is Lys, Glu, Gly, Arg, Met, Leu, His or Asp; Xaa at position76 is Asp or Gin; Xaa at position 79 is Lys or Glu; Xaa at position 80is Glu or Ser; Xaa at position 82 is Glu, Ile, Leu, Tyr or Gin; Xaa atposition 83 is Glu or Gin; Xaa at position 84 is Tyr or Phe; Xaa atposition 86 is Glu or Gin; Xaa at position 87 is Lys or Gin; Xaa atposition 88 is Met, Ile or Leu; Xaa at position 90 is Gin or Glu; Xaa atposition 94 is Val or Ile; Xaa at position 97 is Arg, Asn, Asp, Glu,Gin, Gly or Ser; Xaa at position 98 is Tyr or Phe; Xaa at position 99 isLys, Leu, Tyr, Ile, Met, Phe, Cys, Val or Asn; Xaa at position 103 isAla or Gly; Xaa at position 105 is Leu or Ile; Xaa at position 109 isPhe, Lys, Gly, Met, Ser, Asp, Asn, Glu, Cys, Ala or Arg; Xaa at position112 is Thr or Ser; Xaa at position 113 is Asp, Glu or Met; Xaa atposition 117 is Thr or Ser; Xaa at position 121 is Tyr or Phe; Xaa atposition 127 is Ala or Thr; Xaa at position 142 is Arg or Glu; Xaa atposition 146 is Arg or Gin; Xaa at position 147 is Arg, Glu or Gin; Xaaat position 148 is Asp, Phe, Pro, Val, Glu, His, Trp, Ala, Arg, Leu,Ser, Gin or Gly; Xaa at position 149 is Phe or Val; Xaa at position 150is Arg, Gin or Glu; Xaa at position 151 is Asp, Ser, Ala, Asn, Trp, Val,Gin, Cys, Met, Leu, Arg or Glu; Xaa at position 153 is Leu or lie; Xaaat position 154 is Asn or Asp; Xaa at position 155 is Asn or Lys; Xaa atposition 159 is Pro or Asp; Xaa at position 162 is Glu, Asp or Gin; Xaaat position 165 is Lys, Glu, Gin, Pro, Thr, Ala, Leu, Gly, Asp, Val,His, lie, Met, Trp, Phe, Tyr or Arg; Xaa at position 166 is Arg or Gin;Xaa at position 167 is Tyr, Trp or Cys; Xaa at position 170 is Tyr orHis; Xaa at position 171 is Tyr or Phe; Xaa at position 172 is lie, Leuor Val; Xaa at position 173 is Ser or Ala; Xaa at position 174 is Glu orGin; Xaa at position 182 is Asp or Gin; Xaa at position 183 is Tyr orVal; Xaa at position 184 is Ser or Thr; Xaa at position 185 is Ala orSer; Xaa at position 189 is Thr, Lys or lie; Xaa at position 191 is Lysor Gin; Xaa at position 193 is Asp or Asn; Xaa at position 196 is Gin,Lys, Asn, Asp, Glu, Ala, lie or Arg; Xaa at position 202 is Ala or Val;Xaa at position 203 is Glu, Thr or His; Xaa at position 204 is Met orAla; Xaa at position 206 is Tyr or Phe; Xaa at position 207 is Lys orGin; Xaa at position 209 is Leu or Pro; Xaa at position 210 is Val orlie; Xaa at position 214 is Lys, Ser or Gin; Xaa at position 216 is Glu,Gin, Phe, Val, Tyr or Arg; Xaa at position 220 is Glu, His, Asp, Thr,Tyr, Val, Ser, Gin, Arg, Trp, Met, Ala, Phe, lie, Leu, Cys or Asn; Xaaat position 229 is Arg or Glu; Xaa at position 230 is Ser or Glu; Xaa atposition 231 is Asn or Ser; Xaa at position 236 is Leu or Pro; Xaa atposition 245 is Met or Leu; Xaa at position 247 is Asp or Tyr; Xaa atposition 256 is Gin, Lys or Glu; Xaa at position 257 is Gin, lie, Glu,Cys, Ser, His, Trp or Met; Xaa at position 261 is Gin, Glu or Lys; Xaaat position 264 is Glu or Gin; Xaa at position 268 is Asp or Asn; Xaa atposition 276 is Ser or Ala; Xaa at position 278 is Glu, Asn or Gin; Xaaat position 281 is Gin, Lys or Glu; Xaa at position 282 is Pro or Gly;Xaa at position 284 is Trp or Arg; Xaa at position 287 is Ala or Cys;Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe,Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala; Xaa at position 291is Glu or Gin; Xaa at position 292 is Arg or Gin; Xaa at position 293 isArg, Glu or Gin; Xaa at position 294 is Val or Ala; Xaa at position 296is Leu or lie; Xaa at position 297 is Glu or Gin; Xaa at position 298 isAsp or Gin; Xaa at position 300 is Phe or Tyr; Xaa at position 302 isGlu or Gin; Xaa at position 303 is Phe or Tyr; Xaa at position 305 isLys or Gin; Xaa at position 306 is Gin or Lys; Xaa at position 309 isGin, Lys or Glu; Xaa at position 313 is Lys, Gin or Arg; Xaa at position316 is Lys or Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa atposition 331 is Glu, Asn or Gin; Xaa at position 333 is Ser, Arg, Gly,Lys, Val, Asn, Ala, His, Gin, Thr, Asp, lie, Leu, Cys or Glu; Xaa atposition 334 is Gly, Arg, Lys, lie or Trp; Xaa at position 335 is Ser orAla; Xaa at position 336 is Gly or Ala; Xaa at position 337 is Ala, Valor Gly; Xaa at position 338 is Ser, His, Val, Lys, Ala, Gly, Thr, lie,Glu, Met, Arg, Pro, Asp, Asn or Leu; Xaa at position 339 is Glu, Asn,Gin, lie, Pro, Met, Ser, Ala, Cys, Phe, Val, Leu, Asp, Trp, His or Arg;Xaa at position 341 is Leu or Val; Xaa at position 342 is Ala, Ser orVal; Xaa at position 343 is Val or lie; Xaa at position 344 is Phe orTrp; Xaa at position 345 is Asn or His; Xaa at position 346 is Pro orAla; Xaa at position 350 is Asn or Ser; Xaa at position 351 is Gly orVal; Xaa at position 354 is Met or Leu; Xaa at position 355 is Val, lieor Leu; Xaa at position 359 is Gly or Ala; Xaa at position 362 is Asn orSer; Xaa at position 364 is Ala or Ser; Xaa at position 371 is Ala, Glyor Thr; Xaa at position 374 is Phe or lie; Xaa at position 375 is Lys orArg; Xaa at position 380 is Leu or Gly; Xaa at position 382 is Val, Aspor Leu; Xaa at position 383 is Leu, lie or Val; Xaa at position 384 isLys, Ala or Gly; Xaa at position 385 is Ala or Gly; Xaa at position 389is Trp or Tyr; Xaa at position 391 is Arg, Leu, Glu, Gin or Asp; Xaa atposition 395 is Asp or Cys; Xaa at position 396 is Ala, Leu, Lys, Asn,Gly, lie, Met, Arg, Tyr, Gin or His; Xaa at position 397 is Gly, Arg orAla; Xaa at position 398 is Ser, Gin or Cys; Xaa at position 401 is Ser,His, Pro, Gly, Lys, Val, Arg, lie, Asn, Phe, Thr, Ala, Asp, Met, Gin orGlu; Xaa at position 402 is Lys, Phe, His, Arg, Trp, Gly, Asn, Leu, Tyr,Thr, Val, Met, Pro or Ala; Xaa at position 403 is Asp, Tyr, Trp, Phe orGlu; Xaa at position 405 is Ala or Ser; Xaa at position 409 is Ala orPro; Xaa at position 410 is lie or Val; Xaa at position 411 is Pro orAla; Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg, Gluor Gin; Xaa at position 417 is Ala, Ser or Cys; Xaa at position 418 isLeu or Met; Xaa at position 422 is Met or Val; Xaa at position 426 isThr or Ser; Xaa at position 436 is Asp or Lys; Xaa at position 437 isTyr or Val; Xaa at position 438 is Val or Arg; Xaa at position 440 isVal or Leu; Xaa at position 442 is Gin, Lys or Glu; Xaa at position 445is Cys, Leu or Thr; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu, lie,Gly, Pro, Leu, Phe, Trp or Thr; Xaa at position 448 is Val or Ala; Xaaat position 449 is Gin or Glu; Xaa at position 452 is Gin, Lys or Glu;Xaa at position 453 is Asn or Asp; Xaa at position 454 is Arg, Tyr, Met,Ser, Val, lie, Lys, Phe, Trp, Gin, Gly, His, Asp, Leu, Thr, Pro or Asn;Xaa at position 455 is Val or lie; Xaa at position 457 is Trp or Asn;Xaa at position 459 is Lys, Met, Val, Trp, Gin, lie, Thr, Ser, His, Cys,Tyr, Pro, Asn, Ala, Arg or Glu; Xaa at position 460 is Gly or Ala; Xaaat position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala; Xaa atposition 463 is Ala, Ser or Gly; Xaa at position 464 is Arg, Gly, His,Gin, Thr or Phe; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly,Arg, Thr, His, Cys, Trp, Phe or Leu; Xaa at position 466 is Asp or Arg;Xaa at position 471 is Gin, Lys or Glu; Xaa at position 497 is Asp orGin; Xaa at position 499 is Glu or Gin; Xaa at position 500 is Arg, Ginor Lys; Xaa at position 502 is Arg, Glu or Gin; Xaa at position 509 isLys, Gin or Glu; Xaa at position 517 is Gin, Cys, Asn, Val or Pro; Xaaat position 518 is Glu or Gin; Xaa at position 520 is Lys, Gin or Glu;Xaa at position 525 is Gin or Lys; and Xaa at position 527 is Gin, Lys,Pro, Cys, Glu, Ser, His, Phe or Trp; and having one or more amino acidsubstitutions at positions designated as Xaa in SEQ ID NO: 4 and whereinthe PHI-4 polypeptide has increased insecticidal activity compared toSEQ ID NO: 35.

In some embodiments the nucleic acid molecule encoding a PHI-4polypeptide is a polynucleotide having a nucleotide sequence encoding apolypeptide comprising an amino acid sequence SEQ ID NO: 3, wherein Xaaat position 24 is Asp or Asn; Xaa at position 42 is Asp or Asn; Xaa atposition 43 is Phe or Glu; Xaa at position 46 is Glu or Asn; Xaa atposition 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or Glu; Xaa atposition 82 is Glu, Ile, Leu or Tyr; Xaa at position 97 is Arg, Asn,Asp, Glu, Gin or Gly; Xaa at position 98 is Tyr or Phe; Xaa at position99 is Lys, Leu, Tyr, Ile or Met; Xaa at position 109 is Phe, Lys, Gly,Met, Ser, Asp or Asn; Xaa at position 147 is Arg or Glu; Xaa at position148 is Asp, Phe or Pro; Xaa at position 150 is Arg or Gin; Xaa atposition 151 is Asp, Ser, Ala or Asn; Xaa at position 153 is Leu or Ile;Xaa at position 162 is Glu or Gin; Xaa at position 165 is Lys, Glu orGin; Xaa at position 166 is Arg or Gin; Xaa at position 171 is Tyr orPhe; Xaa at position 174 is Glu or Gin; Xaa at position 182 is Asp orGin; Xaa at position 196 is Gin, Lys, Asn or Asp; Xaa at position 203 isGlu, Thr or His; Xaa at position 206 is Tyr or Phe; Xaa at position 216is Glu or Gin; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Seror Gin; Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gin orLys; Xaa at position 257 is Gin or Ile; Xaa at position 261 is Gin orGlu; Xaa at position 278 is Glu or Asn; Xaa at position 281 is Gin, Lysor Glu; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr orAsp; Xaa at position 293 is Arg, Glu or Gin; Xaa at position 313 is Lysor Gin; Xaa at position 328 is Lys, Glu or Gin; Xaa at position 333 isSer, Gly, Lys, Val or Asn; Xaa at position 334 is Gly, Arg, Lys or Ile;Xaa at position 336 is Gly or Ala; Xaa at position 338 is Ser, His, Val,Lys or Ala; Xaa at position 339 is Glu, Asn, Ile or Pro; Xaa at position343 is Val or Ile; Xaa at position 346 is Pro or Ala; Xaa at position355 is Val or Ile; Xaa at position 359 is Gly or Ala; Xaa at position391 is Arg, Glu or Gin; Xaa at position 396 is Ala, Leu, Lys, Asn orGly; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val or Arg; Xaa atposition 402 is Lys, Phe, His, Arg, Gly, Trp, Thr, Asn, Tyr or Met; Xaaat position 403 is Asp or Tyr; Xaa at position 411 is Pro or Ala; Xaa atposition 412 is Pro or Ala; Xaa at position 416 is Arg or Glu; Xaa atposition 417 is Ala or Ser; Xaa at position 418 is Leu or Met; Xaa atposition 426 is Thr or Ser; Xaa at position 440 is Val or Leu; Xaa atposition 447 is Asp, Lys, Tyr, Ser, Glu or Ile; Xaa at position 452 isGin, Lys or Glu; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, Ile,Lys, Phe, Trp or Gin; Xaa at position 455 is Val or Ile; Xaa at position459 is Lys, Met, Val, Trp, Gin, Ile or Tyr; Xaa at position 461 is Thror Ser; Xaa at position 462 is Gly or Ala; Xaa at position 463 is Ala orSer; Xaa at position 464 is Arg, Gly or His; Xaa at position 465 is Lys,Asn, Val, Met, Pro, Gly or Arg; Xaa at position 471 is Gin, Lys or Glu;Xaa at position 500 is Arg or Gin; Xaa at position 509 is Lys or Gin;Xaa at position 520 is Lys, Gin or Glu; and Xaa at position 527 is Gin,Lys, Pro, Cys or Glu; and having one or more amino acid substitutions atpositions designated as Xaa in SEQ ID NO: 3 and wherein the PHI-4polypeptide has increased insecticidal activity compared to SEQ ID NO:35.

In some embodiments exemplary nucleic acid molecules encode a PHI-4 ofSEQ ID NO: 51-819 as well as amino acid substitutions, amino aciddeletions, insertions and fragments thereof and combinations thereof.

In some embodiments the nucleic acid molecules encode a PHI-4polypeptide of Table 3, Table 4, Table 5, Table 6, Table 7, Megatable 1,and Megatable 2 and combinations of the amino acid substitutions thereofand amino acid deletions and/or insertions thereof.

Also provided are nucleic acid molecules that encode transcriptionand/or translation products that are subsequently spliced to ultimatelyproduce functional PHI-4. Splicing can be accomplished in vitro or invivo, and can involve cis- or trans-splicing. The substrate for splicingcan be polynucleotides (e.g., RNA transcripts) or polypeptides. Anexample of cis-splicing of a polynucleotide is where an intron insertedinto a coding sequence is removed and the two flanking exon regions arespliced to generate a PHI-4 encoding sequence. An example of transsplicing would be where a polynucleotide is encrypted by separating thecoding sequence into two or more fragments that can be separatelytranscribed and then spliced to form the full-length pesticidal encodingsequence. The use of a splicing enhancer sequence, which can beintroduced into a construct, can facilitate splicing either in cis ortrans-splicing of polypeptides (U.S. Pat. Nos. 6,365,377 and 6,531,316).Thus, in some embodiments the polynucleotides do not directly encode afull-length PHI-4, but rather encode a fragment or fragments of a PHI-4.These polynucleotides can be used to express a functional PHI-4 througha mechanism involving splicing, where splicing can occur at the level ofpolynucleotide (e.g., intron/exon) and/or polypeptide (e.g.,intein/extein). This can be useful, for example, in controllingexpression of pesticidal activity, since functional pesticidalpolypeptide will only be expressed if all required fragments areexpressed in an environment that permits splicing processes to generatefunctional product. In another example, introduction of one or moreinsertion sequences into a polynucleotide can facilitate recombinationwith a low homology polynucleotide; use of an intron or intein for theinsertion sequence facilitates the removal of the intervening sequence,thereby restoring function of the encoded variant.

Nucleic acid molecules that are fragments of these nucleic acidsequences encoding PHI-4 are also encompassed by the embodiments. By“fragment” is intended a portion of the nucleic acid sequence encoding aPHI-4. A fragment of a nucleic acid sequence may encode a biologicallyactive portion of a PHI-4 polypeptide or it may be a fragment that canbe used as a hybridization probe or PCR primer using methods disclosedbelow. Nucleic acid molecules that are fragments of a nucleic acidsequence encoding a PHI-4 comprise at least about 50, 100, 200, 300,400, 500, 600 or 700, contiguous nucleotides, or up to the number ofnucleotides present in a full-length nucleic acid sequence encoding aPHI-4 disclosed herein, depending upon the intended use. By “contiguous”nucleotides is intended nucleotide residues that are immediatelyadjacent to one another. Fragments of the nucleic acid sequences of theembodiments will encode protein fragments that retain the biologicalactivity of the PHI-4 and, hence, retain insecticidal activity. By“retains activity” is intended that the fragment will encode apolypeptide having at least about 30%, at least about 50%, at leastabout 70%, 80%, 90%, 95% or higher of the insecticidal activity of thefull-length PHI-4. In one embodiment, the insecticidal activity isLepodoptera activity. In another embodiment, the insecticidal activityis Hemiptera activity.

In some embodiments a fragment of a nucleic acid sequence encoding aPHI-4 encoding a biologically active portion of a protein will encode atleast about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, or 250,contiguous amino acids, or up to the total number of amino acids presentin a full-length PHI-4 of the embodiments. In some embodiments, thefragment is an N-terminal or a C-terminal truncation of at least about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25 or more amino acids relative to SEQ ID NO: 35, SEQ ID NOs: 51-819 orvariants thereof, e.g., by proteolysis, insertion of a start codon,deletion of the codons encoding the deleted amino acids with theconcomitant insertion of a stop codon or by insertion of a stop codon inthe coding sequence. In some embodiments, the fragments encompassedherein result from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, or more amino acids relative to SEQ IDNO: 35, SEQ ID NOs: 51-819 or variants thereof, e.g., by proteolysis orby insertion of a start codon in the coding sequence.

In some embodiments the PHI-4 are encoded by a nucleic acid sequencesufficiently identical to the nucleic acid sequence of SEQ ID NO: 1, SEQID NO: 7, SEQ ID NO: 11, SEQ ID NOS: 24-30. By “sufficiently identical”is intended an amino acid or nucleic acid sequence that has at leastabout 60% or 65% sequence identity, about 70% or 75% sequence identity,about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity comparedto a reference sequence using one of the alignment programs describedherein using standard parameters. In some embodiments the sequencehomology identity is against the full length sequence of thepolynucleotide encoding a PHI-4 or against the full length sequence of aPHI-4 polypeptide. In some embodiments the polynucleotide encoding thePHI-4 has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 1, SEQID NO: 7, SEQ ID NO: 11, SEQ ID NOS: 24-30. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleic acid sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning, and the like.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence (e.g., across the entirety of SEQ IDNO: 35 or across the entirety of one of SEQ ID NO: 1, SEQ ID NO: 7, SEQID NO: 11, SEQ ID NOS: 24-30). The percent identity between twosequences can be determined using techniques similar to those describedbelow, with or without allowing gaps. In calculating percent identity,typically exact matches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul, (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul, et al., (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleic acid sequences homologous topesticidal-like nucleic acid molecules. BLAST protein searches can beperformed with the BLASTX program, score=50, wordlength=3, to obtainamino acid sequences homologous to pesticidal protein molecules. Toobtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST2.0) can be utilized as described in Altschul, et al., (1997) NucleicAcids Res. 25:3389. Alternatively, PSI-Blast can be used to perform aniterated search that detects distant relationships between molecules.See, Altschul, et al., (1997) supra. When utilizing BLAST, Gapped BLAST,and PSI-Blast programs, the default parameters of the respectiveprograms (e.g., BLASTX and BLASTN) can be used. Alignment may also beperformed manually by inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins, et al.,(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNX®module of the Vector NTI® Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12 and a gap penalty of 4can be used.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Needleman and Wunsch,(1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software todetermine sequence identity or similarity using the following defaultparameters: % identity and % similarity for a nucleic acid sequenceusing GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmpiiscoring matrix; % identity or % similarity for an amino acid sequenceusing GAP weight of 8 and length weight of 2, and the BLOSUM62 scoringprogram. Equivalent programs may also be used. By “equivalent program”is intended any sequence comparison program that, for any two sequencesin question, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The embodiments also encompass nucleic acid molecules encoding variantsof PHI-4 polypeptide. “Variants” of the PHI-4 polypeptide encodingnucleic acid sequences include those sequences that encode the PHI-4polypeptides disclosed herein but that differ conservatively because ofthe degeneracy of the genetic code as well as those that aresufficiently identical as discussed above. Naturally occurring allelicvariants can be identified with the use of well-known molecular biologytechniques, such as polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleic acid sequences alsoinclude synthetically derived nucleic acid sequences that have beengenerated, for example, by using site-directed mutagenesis but whichstill encode the PHI-4 polypeptides disclosed as discussed below.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences thereby leading tochanges in the amino acid sequence of the encoded PHI-4 polypeptides,without altering the biological activity of the proteins. Thus, variantnucleic acid molecules can be created by introducing one or morenucleotide substitutions, nucleotide additions and/or nucleotidedeletions into the corresponding nucleic acid sequence disclosed herein,such that one or more amino acid substitutions, amino acid additions oramino acid deletions are introduced into the encoded protein. Mutationscan be introduced by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Such variant nucleic acidsequences are also encompassed by the present invention.

Alternatively, variant nucleic acid sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional pesticidal polypeptide homologues and fragmentsthereof with desired properties. A variety of such reactions are known,including those developed by the inventors and their co-workers. Methodsfor producing a variant of any nucleic acid listed herein comprisingrecursively recombining such polynucleotide with a second (or more)polynucleotide, thus forming a library of variant polynucleotides arealso embodiments of the disclosure, as are the libraries produced, thecells comprising the libraries, and any recombinant polynucleotideproduces by such methods. Additionally, such methods optionally compriseselecting a variant polynucleotide from such libraries based onpesticidal activity, as is wherein such recursive recombination is donein vitro or in vivo.

A variety of diversity generating protocols, including nucleic acidrecursive recombination protocols are available and fully described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein, or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.pesticidal activity, or, such activity at a desired pH, etc. This caninclude identifying any activity that can be detected, for example, inan automated or automatable format, by any of the assays in the art,see, e.g., discussion of screening of insecticidal activity, infra. Avariety of related (or even unrelated) properties can be evaluated, inserial or in parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences, e.g., those coding forpolypeptides having pesticidal activity, or fragments thereof, are foundin the following publications and the references cited therein: Soong,et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) TumorTargeting 4:1-4; Ness et al. (1999) Nat Biotechnol 17:893-896; Chang etal. (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999) CurrOpin Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al.,(1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733;Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) NatBiotechnol 14:315-319; Gates, et al., (1996) J Mol Biol 255:373-386;Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia ofMolecular Biology. VCH Publishers, New York. pp. 447-457; Crameri andStemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene,164:49-53; Stemmer, (1995) Science 270:1510; Stemmer, (1995)Bio/Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 andStemmer, (1994) PNAS USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling, et al., (1997) Anal Biochem254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith,(1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science229:1193-1201; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) “Theefficiency of oligonucleotide directed mutagenesis” in Nucleic Acids &Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin));mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass,et al., (1988) Science 242:240-245); oligonucleotide-directedmutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500;Zoller and Smith, (1987) Methods Enzymol 154:329-350; Zoller and Smith,(1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modified DNAmutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764; Taylor,et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye and Eckstein(1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988) Nucl AcidsRes 16:791-802 and Sayers, et al., (1988) Nucl Acids Res 16: 803-814);mutagenesis using gapped duplex DNA (Kramer, et al., (1984) Nucl AcidsRes 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol 154:350-367;Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, et al., (1988)Nucl Acids Res 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, etal., (1984) Cell 38:879-887), mutagenesis using repair-deficient hoststrains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter,(1987) Methods in Enzymol 154:382-403), deletion mutagenesis(Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115),restriction-selection and restriction-purification (Wells, et al.,(1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total genesynthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar andKhorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene34:315-323 and Grundstrom, et al., (1985) Nucl Acids Res 13:3305-3316),double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 andArnold, (1993) Curr Opin Biotech 4:450-455). Additional details on manyof the above methods can be found in Methods Enzymol Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following US Patents, PCT Publications and Applications andEPO Publications: U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476,5,817,483, 5,824,514, 5,976,862, 5,605,793, 5,811,238, 5,830,721,5,834,252, 5,837,458, WO 1995/22625, WO 1996/33207, WO 1997/20078, WO1997/35966, WO 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/41368,EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, WO 1998/31837, WO1998/27230, WO 1998/27230, WO 2000/00632, WO 2000/09679, WO 1998/42832,WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO2000/18906, WO 2000/04190, WO 2000/42561, WO 2000/42559, WO 2000/42560,WO 2001/23401, and PCT/US01/06775.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from other organisms, particularly otherbacteria. In this manner, methods such as PCR, hybridization and thelike can be used to identify such sequences based on their sequencehomology to the sequences set forth herein. Sequences that are selectedbased on their sequence identity to the entire sequences set forthherein or to fragments thereof are encompassed by the embodiments. Suchsequences include sequences that are orthologs of the disclosedsequences. The term “orthologs” refers to genes derived from a commonancestral gene and which are found in different species as a result ofspeciation. Genes found in different species are considered orthologswhen their nucleotide sequences and/or their encoded protein sequencesshare substantial identity as defined elsewhere herein. Functions oforthologs are often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), hereinafter “Sambrook”. See also, Innis, et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

To identify potential PHI-4 polypeptides from bacterial collections, thebacterial cell lysates can be screened with antibodies generated againsta PHI-4 polypeptide using Western blotting and/or ELISA methods. Thistype of assays can be performed in a high throughput fashion. Positivesamples can be further analyzed by various techniques such as antibodybased protein purification and identification. Methods of generatingantibodies are well known in the art as discussed infra.

Alternatively, mass spectrometry based protein identification method canbe used to identify homologs of a PHI-4 polypeptide using protocols inthe literatures (Patterson, (1998), 10(22):1-24, Current Protocol inMolecular Biology published by John Wiley & Son Inc). Specifically,LC-MS/MS based protein identification method is used to associate the MSdata of given cell lysate or desired molecular weight enriched samples(excised from SDS-PAGE gel of relevant molecular weight bands to a PHI-4polypeptide) with sequence information of a PHI-4 polypeptide and itshomologs. Any match in peptide sequences indicates the potential ofhaving the homologous proteins in the samples. Additional techniques(protein purification and molecular biology) can be used to isolate theprotein and identify the sequences of the homologs.

In hybridization methods, all or part of the pesticidal nucleic acidsequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, (2001), supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides, and may be labeledwith a detectable group such as 32P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known PHI-4 polypeptide-encodingnucleic acid sequence disclosed herein. Degenerate primers designed onthe basis of conserved nucleotides or amino acid residues in the nucleicacid sequence or encoded amino acid sequence can additionally be used.The probe typically comprises a region of nucleic acid sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutivenucleotides of nucleic acid sequence encoding a PHI-4 polypeptide of thedisclosure or a fragment or variant thereof. Methods for the preparationof probes for hybridization are generally known in the art and aredisclosed in Sambrook and Russell, (2001), supra, herein incorporated byreference.

For example, an entire nucleic acid sequence, encoding a PHI-4polypeptide, disclosed herein, or one or more portions thereof, may beused as a probe capable of specifically hybridizing to correspondingnucleic acid sequences encoding PHI-4 polypeptide-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length, or at least about 20nucleotides in length. Such probes may be used to amplify correspondingpesticidal sequences from a chosen organism by PCR. This technique maybe used to isolate additional coding sequences from a desired organismor as a diagnostic assay to determine the presence of coding sequencesin an organism. Hybridization techniques include hybridization screeningof plated DNA libraries (either plaques or colonies; see, for example,Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284:Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with ≥90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3or 4° C. lower than the thermal melting point (Tm); moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10°C. lower than the thermal melting point (Tm); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point (Tm). Using the equation,hybridization and wash compositions, and desired Tm, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen, (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel, et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See, Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.).

Proteins and Variants and Fragments Thereof

PHI-4 polypeptides are encompassed by the disclosure. By “PHI-4polypeptide” or “PHI-4 protein” as used herein interchangeably isintended a polypeptide that has increased insecticidal activity againstone or more insect pests of the Lepidoptera and/or Coleoptera orderscompared to the protein of SEQ ID NO: 35, and is sufficiently identicalto the protein of SEQ ID NO: 35. A variety of PHI-4 polypeptides arecontemplated.

The Western Corn Rootworm active protein AXMI-205 (SEQ ID NO: 35)encoded by the polynucleotide of SEQ ID NO: 34 was identified from theChromobacterium Strain ATX 2024 (US20110023184). Synthetic genesencoding AXMI-205; AXMI-205 variants having a truncation of the last 10and 20 amino acids from the C-terminus (SEQ ID NO: 36 and SEQ ID NO:37); and alanine scanning at every other residue from residue 307-536 ofAXMI-205 (SEQ ID NO: 35), with S307A, D315A, V317A, S349A, G351A, K353A,V355A, D395A, G399A, W407A, G419A, P435A, S443A, K465A, V467A, F483A,P487A, S495A, D497A, E499A, K509A, and 1513A identified as having WCRWactivity; are disclosed in US20110023184. AXMI-205 variants evo 24(E499A); evo25 (V467A—SEQ ID NO: 41); evo30 (V467L—SEQ ID NO: 42);PMlibl PoollG2_p2al 1 (S468L—SEQ ID NO: 45); PMlibl PoollG2_plcl(V467T—SEQ ID NO: 46), PMlibl PoollG21pla4 (R464N—SEQ ID NO: 47), evo34(E86T—SEQ ID NO: 43) and evo35 (Q517R—SEQ ID NO: 44) having increasedinsecticidal activity are disclosed in WO2013/016617.

As used herein, the terms “protein,” “peptide molecule” or “polypeptide”includes any molecule that comprises five or more amino acids. It iswell known in the art that protein, peptide or polypeptide molecules mayundergo modification, including post-translational modifications, suchas, but not limited to, disulfide bond formation, glycosylation,phosphorylation or oligomerization. Thus, as used herein, the terms“protein,” “peptide molecule” or “polypeptide” includes any protein thatis modified by any biological or non-biological process. The terms“amino acid” and “amino acids” refer to all naturally occurring L-aminoacids.

A “recombinant protein” is used to refer to a protein that is no longerin its natural environment, for example in vitro or in a recombinantbacterial or plant host cell. A PHI-4 polypeptide that is substantiallyfree of cellular material includes preparations of protein having lessthan about 30%, 20%, 10% or 5% (by dry weight) of non-pesticidal protein(also referred to herein as a “contaminating protein”).

By “improved activity” or “increased activity” is intended an increaseof at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100%, at least about 110%,at least about 120%, at least about 130%, at least about 140%, at leastabout 150%, at least about 160%, at least about 170%, at least about180%, at least about 190%, at least about 200%, at least about 210% atleast about 220%, at least about 230%, at least about 240%, at leastabout 250%, at least about 260%, at least about 270%, at least about280%, at least about 290%, at least about 300%, at least about 310%, atleast about 320%, at least about 330%, at least about 340%, at leastabout 350%, at least about 360%, at least about 370%, at least about380%, at least about 390%, at least about 400%, at least about 410%, atleast about 420%, at least about 430%, at least about 440%, at leastabout 450%, at least about 460%, at least about 470%, at least about480%, at least about 490%, at least about 500%, at least about 510%, atleast about 520%, at least about 530%, at least about 540%, at leastabout 550%, at least about 560%, at least about 570%, at least about580%, at least about 590%, at least about 600%, at least about 650%, atleast about 700%, at least about 750%, at least about 800%, at leastabout 850%, at least about 900%, at least about 950%, at least about1000% or higher, or at least about 1.1-fold, at least about 1.2-fold, atleast about 1.3-fold, at least about 1.4-fold, or at least about1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at leastabout 1.8-fold, at least about 1.9-fold, at least about 2-fold, at leastabout 2.1-fold, at least about 2.2-fold, at least about 2.3-fold, atleast about 2.4-fold, at least about 2.5-fold, at least about 2.6-fold,at least about 2.7-fold, at least about 2.8-fold, at least about2.9-fold, at least about 3-fold, at least about 3.1-fold, at least about3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at leastabout 3.5-fold, at least about 3.6-fold, at least about 3.7-fold, atleast about 3.8-fold, at least about 3.9-fold, at least about 4-fold, atleast about 4.1-fold, at least about 4.2-fold, at least about 4.3-fold,at least about 4.4-fold, at least about 4.5-fold, at least about4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at leastabout 4.9-fold, at least about 5-fold, at least about 5.1-fold, at leastabout 5.2-fold, at least about 5.3-fold, at least about 5.4-fold, atleast about 5.5-fold, at least about 5.6-fold, at least about 5.7-fold,at least about 5.8-fold, at least about 5.9-fold, at least about 6-fold,at least about 6.1-fold, at least about 6.2-fold, at least about6.3-fold, at least about 6.4-fold, at least about 6.5-fold, at leastabout 6.6-fold, at least about 6.7-fold, at least about 6.8-fold, atleast about 6.9-fold, at least about 7-fold, at least about 7.1-fold, atleast about 7.2-fold, at least about 7.3-fold, at least about 7.4-fold,at least about 7.5-fold, at least about 7.6-fold, at least about7.7-fold, at least about 7.8-fold, at least about 7.9-fold, at leastabout 8-fold, at least about 8.1-fold, at least about 8.2-fold, at leastabout 8.3-fold, at least about 8.4-fold, at least about 8.5-fold, atleast about 8.6-fold, at least about 8.7-fold, at least about 8.8-fold,at least about 8.9-fold, at least about 9-fold, at least about 9.1-fold,at least about 9.2-fold, at least about 9.3-fold, at least about9.4-fold, at least about 9.5-fold, at least about 9.6-fold, at leastabout 9.7-fold, at least about 9.8-fold, at least about 9.9-fold, atleast about 10-fold, or higher increase in the pesticidal activity ofthe variant protein relative to the pesticidal activity of AXMI-205 (SEQID NO: 35).

In some embodiments, the improvement consists of a decrease in the EC50of at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 100%, at least about 110%,at least about 120%, at least about 130%, at least about 140%, at leastabout 150%, at least about 160%, at least about 170%, at least about180%, at least about 190%, at least about 200%, at least about 210% atleast about 220%, at least about 230%, at least about 240%, at leastabout 250%, at least about 260%, at least about 270%, at least about280%, at least about 290%, at least about 300%, at least about 310%, atleast about 320%, at least about 330%, at least about 340%, at leastabout 350%, at least about 360%, at least about 370%, at least about380%, at least about 390%, at least about 400%, at least about 410%, atleast about 420%, at least about 430%, at least about 440%, at leastabout 450%, at least about 460%, at least about 470%, at least about480%, at least about 490%, at least about 500%, at least about 510%, atleast about 520%, at least about 530%, at least about 540%, at leastabout 550%, at least about 560%, at least about 570%, at least about580%, at least about 590%, at least about 600%, at least about 650%, atleast about 700%, at least about 750%, at least about 800%, at leastabout 850%, at least about 900%, at least about 950%, at least about1000% or higher, or at least about 1.1-fold, at least about 1.2-fold, atleast about 1.3-fold, at least about 1.4-fold, or at least about1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at leastabout 1.8-fold, at least about 1.9-fold, at least about 2-fold, at leastabout 2.1-fold, at least about 2.2-fold, at least about 2.3-fold, atleast about 2.4-fold, at least about 2.5-fold, at least about 2.6-fold,at least about 2.7-fold, at least about 2.8-fold, at least about2.9-fold, at least about 3-fold, at least about 3.1-fold, at least about3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at leastabout 3.5-fold, at least about 3.6-fold, at least about 3.7-fold, atleast about 3.8-fold, at least about 3.9-fold, at least about 4-fold, atleast about 4.1-fold, at least about 4.2-fold, at least about 4.3-fold,at least about 4.4-fold, at least about 4.5-fold, at least about4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at leastabout 4.9-fold, at least about 5-fold, at least about 5.1-fold, at leastabout 5.2-fold, at least about 5.3-fold, at least about 5.4-fold, atleast about 5.5-fold, at least about 5.6-fold, at least about 5.7-fold,at least about 5.8-fold, at least about 5.9-fold, at least about 6-fold,at least about 6.1-fold, at least about 6.2-fold, at least about6.3-fold, at least about 6.4-fold, at least about 6.5-fold, at leastabout 6.6-fold, at least about 6.7-fold, at least about 6.8-fold, atleast about 6.9-fold, at least about 7-fold, at least about 7.1-fold, atleast about 7.2-fold, at least about 7.3-fold, at least about 7.4-fold,at least about 7.5-fold, at least about 7.6-fold, at least about7.7-fold, at least about 7.8-fold, at least about 7.9-fold, at leastabout 8-fold, at least about 8.1-fold, at least about 8.2-fold, at leastabout 8.3-fold, at least about 8.4-fold, at least about 8.5-fold, atleast about 8.6-fold, at least about 8.7-fold, at least about 8.8-fold,at least about 8.9-fold, at least about 9-fold, at least about 9.1-fold,at least about 9.2-fold, at least about 9.3-fold, at least about9.4-fold, at least about 9.5-fold, at least about 9.6-fold, at leastabout 9.7-fold, at least about 9.8-fold, at least about 9.9-fold, atleast about 10-fold, or greater reduction in the EC50 of the PHI-4polypeptide relative to the pesticidal activity of AXMI-205 (SEQ ID NO:35).

In some embodiments the EC50 of the PHI-4 polypeptide is <100 ppm, <90ppm, <80 ppm, <70 ppm, <60 ppm, <50 ppm, <45 ppm, <40 ppm, <35 ppm, <30ppm, <25 ppm, <20 ppm, <19 ppm, <18 ppm, <17 ppm, <16 ppm, <15 ppm, <14ppm, <13 ppm, <12 ppm, <11 ppm, <10 ppm, <9 ppm, <8 ppm, <7 ppm, <6 ppm,<5 ppm, <4 ppm, <3 ppm, <2 ppm, <1 ppm, <0.9 ppm, <0.8 ppm, <0.7 ppm,<0.6 ppm, <0.5 ppm, <0.4 ppm, <0.3 ppm, <0.2 ppm, <0.1 ppm,

In some embodiments, the improvement consists of an increase in the MeanFAE Index of at least about 10%, at least about 15%, at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 100%, at leastabout 110%, at least about 120%, at least about 130%, at least about140%, at least about 150%, at least about 160%, at least about 170%, atleast about 180%, at least about 190%, at least about 200%, at leastabout 210% at least about 220%, at least about 230%, at least about240%, at least about 250%, at least about 260%, at least about 270%, atleast about 280%, at least about 290%, at least about 300%, at leastabout 310%, at least about 320%, at least about 330%, at least about340%, at least about 350%, at least about 360%, at least about 370%, atleast about 380%, at least about 390%, at least about 400%, at leastabout 410%, at least about 420%, at least about 430%, at least about440%, at least about 450%, at least about 460%, at least about 470%, atleast about 480%, at least about 490%, at least about 500%, at leastabout 510%, at least about 520%, at least about 530%, at least about540%, at least about 550%, at least about 560%, at least about 570%, atleast about 580%, at least about 590%, at least about 600%, at leastabout 650%, at least about 700%, at least about 750%, at least about800%, at least about 850%, at least about 900%, at least about 950%, atleast about 1000% or higher, or at least about 1.1-fold, at least about1.2-fold, at least about 1.3-fold, at least about 1.4-fold, or at leastabout 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, atleast about 1.8-fold, at least about 1.9-fold, at least about 2-fold, atleast about 2.1-fold, at least about 2.2-fold, at least about 2.3-fold,at least about 2.4-fold, at least about 2.5-fold, at least about2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at leastabout 2.9-fold, at least about 3-fold, at least about 3.1-fold, at leastabout 3.2-fold, at least about 3.3-fold, at least about 3.4-fold, atleast about 3.5-fold, at least about 3.6-fold, at least about 3.7-fold,at least about 3.8-fold, at least about 3.9-fold, at least about 4-fold,at least about 4.1-fold, at least about 4.2-fold, at least about4.3-fold, at least about 4.4-fold, at least about 4.5-fold, at leastabout 4.6-fold, at least about 4.7-fold, at least about 4.8-fold, atleast about 4.9-fold, at least about 5-fold, at least about 5.1-fold, atleast about 5.2-fold, at least about 5.3-fold, at least about 5.4-fold,at least about 5.5-fold, at least about 5.6-fold, at least about5.7-fold, at least about 5.8-fold, at least about 5.9-fold, at leastabout 6-fold, at least about 6.1-fold, at least about 6.2-fold, at leastabout 6.3-fold, at least about 6.4-fold, at least about 6.5-fold, atleast about 6.6-fold, at least about 6.7-fold, at least about 6.8-fold,at least about 6.9-fold, at least about 7-fold, at least about 7.1-fold,at least about 7.2-fold, at least about 7.3-fold, at least about7.4-fold, at least about 7.5-fold, at least about 7.6-fold, at leastabout 7.7-fold, at least about 7.8-fold, at least about 7.9-fold, atleast about 8-fold, at least about 8.1-fold, at least about 8.2-fold, atleast about 8.3-fold, at least about 8.4-fold, at least about 8.5-fold,at least about 8.6-fold, at least about 8.7-fold, at least about8.8-fold, at least about 8.9-fold, at least about 9-fold, at least about9.1-fold, at least about 9.2-fold, at least about 9.3-fold, at leastabout 9.4-fold, at least about 9.5-fold, at least about 9.6-fold, atleast about 9.7-fold, at least about 9.8-fold, at least about 9.9-fold,at least about 10-fold, or higher increase in the Mean FAE Index of thePHI-4 polypeptide relative to the pesticidal activity of AXMI-205 (SEQID NO: 35).

In some embodiments, the improvement consists of an increase in the MeanDeviation Score of at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 100%,at least about 110%, at least about 120%, at least about 130%, at leastabout 140%, at least about 150%, at least about 160%, at least about170%, at least about 180%, at least about 190%, at least about 200%, atleast about 210% at least about 220%, at least about 230%, at leastabout 240%, at least about 250%, at least about 260%, at least about270%, at least about 280%, at least about 290%, at least about 300%, atleast about 310%, at least about 320%, at least about 330%, at leastabout 340%, at least about 350%, at least about 360%, at least about370%, at least about 380%, at least about 390%, at least about 400%, atleast about 410%, at least about 420%, at least about 430%, at leastabout 440%, at least about 450%, at least about 460%, at least about470%, at least about 480%, at least about 490%, at least about 500%, atleast about 510%, at least about 520%, at least about 530%, at leastabout 540%, at least about 550%, at least about 560%, at least about570%, at least about 580%, at least about 590%, at least about 600%, atleast about 650%, at least about 700%, at least about 750%, at leastabout 800%, at least about 850%, at least about 900%, at least about950%, at least about 1000% or higher, or at least about 1.1-fold, atleast about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold,or at least about 1.5-fold, at least about 1.6-fold, at least about1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at leastabout 2-fold, at least about 2.1-fold, at least about 2.2-fold, at leastabout 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, atleast about 2.6-fold, at least about 2.7-fold, at least about 2.8-fold,at least about 2.9-fold, at least about 3-fold, at least about 3.1-fold,at least about 3.2-fold, at least about 3.3-fold, at least about3.4-fold, at least about 3.5-fold, at least about 3.6-fold, at leastabout 3.7-fold, at least about 3.8-fold, at least about 3.9-fold, atleast about 4-fold, at least about 4.1-fold, at least about 4.2-fold, atleast about 4.3-fold, at least about 4.4-fold, at least about 4.5-fold,at least about 4.6-fold, at least about 4.7-fold, at least about4.8-fold, at least about 4.9-fold, at least about 5-fold, at least about5.1-fold, at least about 5.2-fold, at least about 5.3-fold, at leastabout 5.4-fold, at least about 5.5-fold, at least about 5.6-fold, atleast about 5.7-fold, at least about 5.8-fold, at least about 5.9-fold,at least about 6-fold, at least about 6.1-fold, at least about 6.2-fold,at least about 6.3-fold, at least about 6.4-fold, at least about6.5-fold, at least about 6.6-fold, at least about 6.7-fold, at leastabout 6.8-fold, at least about 6.9-fold, at least about 7-fold, at leastabout 7.1-fold, at least about 7.2-fold, at least about 7.3-fold, atleast about 7.4-fold, at least about 7.5-fold, at least about 7.6-fold,at least about 7.7-fold, at least about 7.8-fold, at least about7.9-fold, at least about 8-fold, at least about 8.1-fold, at least about8.2-fold, at least about 8.3-fold, at least about 8.4-fold, at leastabout 8.5-fold, at least about 8.6-fold, at least about 8.7-fold, atleast about 8.8-fold, at least about 8.9-fold, at least about 9-fold, atleast about 9.1-fold, at least about 9.2-fold, at least about 9.3-fold,at least about 9.4-fold, at least about 9.5-fold, at least about9.6-fold, at least about 9.7-fold, at least about 9.8-fold, at leastabout 9.9-fold, at least about 10-fold, or higher increase in the MeanDeviation Score of the PHI-4 polypeptide relative to the pesticidalactivity of AXMI-205 (SEQ ID NO: 35).

In some embodiments the improved activity of the PHI-4 polypeptide isrelative to the pesticidal activity of AXMI-205(evo25) (SEQ ID NO: 41),AXMI-205(evo30) (SEQ ID NO: 42), Axmi205 PMlibl PoollG2_p2al 1 (mutationS468L; SEQ ID NO: 45), Axmi205 PMlibl PoollG2_plcl (mutation V467T; SEQID NO: 46), Axmi205 PMlibl PoollG2_pla4 (mutation R464N; SEQ ID NO: 47),AXMI-205(evo34) (SEQ ID NO: 43) or AXMI-205(evo35) (SEQ ID NO: 44).

“Mean FAE Index” (MFI) refers to the mean of multiple FAEGN anarithmetic mean of FAEGN. As used herein, the “Mean Deviation Score”refers to the arithmetic mean of multiple Deviation Scores.

In some embodiments the PHI-4 polypeptides have increased insecticidalactivity against one or more insect pests of the order Coleoptera.

In some embodiments the PHI-4 polypeptides have increased insecticidalactivity against Diabrotica virgifera larvae—Western Corn Root Worm(WCRW).

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to aPHI-4 polypeptide and that exhibit insecticidal activity. “Fragments” or“biologically active portions” include polypeptide fragments comprisingamino acid sequences sufficiently identical to the amino acid sequenceset forth in SEQ ID NO: 35 and SEQ ID NOs: 51-819 and that exhibitinsecticidal activity. A biologically active portion of a PHI-4polypeptide can be a polypeptide that is, for example, 10, 25, 50, 100,150, 200, 250, 300, 350, 400, 450, 500, 516, 517, 518, 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534 or 535amino acids in length. Such biologically active portions can be preparedby recombinant techniques and evaluated for insecticidal activity. Asused here, a fragment comprises at least 8 contiguous amino acids of aPHI-4 polypeptide. In some embodiments a fragment comprises at least 8contiguous amino acids of SEQ ID NO: 2 or SEQ ID NOs: 51-819. In someembodiments a fragment comprises at least 8 contiguous amino acids ofSEQ ID NO: 2 or SEQ ID NOs: 51-819. The embodiments encompass otherfragments, however, such as any fragment in the protein greater thanabout 10, 20, 30, 50, 100, 150, 200, 250 or more amino acids.

In some embodiments, the fragment is an N-terminal and/or a C-terminaltruncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25 or more amino acids relative to SEQ IDNO: 2, SEQ ID NO: 35 or SEQ ID NOs: 51-819 or variants thereof e.g., byproteolysis, by insertion of a start codon, by deletion of the codonsencoding the deleted amino acids and concomitant insertion of a startcodon and/or insertion of a stop codon. In some embodiments, thefragments encompassed herein result from the removal of the C-terminal1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acids relative to SEQ ID NO:35, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819, andvariants thereof by proteolysis or by insertion of a start codon, bydeletion of the codons encoding the deleted amino acids and concomitantinsertion of a start codon. In particular embodiments the proteolyticcleavage site is between Lys at 520 and Ser at 521 Ser or Lys at 313 andVal at 314 of SEQ ID NO: 35 or variants thereof. It is well known in theart that polynucleotide encoding the truncated PHI-4 polypeptide can beengineered to add a start codon at the N-terminus such as ATG encodingmethionine or methionine followed by an alanine. It is also well knownin the art that depending on what host the PHI-4 polypeptide isexpressed in the methionine may be partially of completed processed off.

In some embodiments fragments, biologically active portions, of SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819 as well as aminoacid substitutions, amino acid deletions and/or insertions thereof arealso provided, and may be used to practice the methods of thedisclosure.

In some embodiments PHI-4 polypeptides are provided having one or moreamino acid substitution compared to AXMI-205 (SEQ ID NO: 35). In someembodiments PHI-4 polypeptides are provided having amino acidsubstitutions at solvent exposed surface residues to modify the proteincharacteristics of AXMI-205 (SEQ ID NO: 35), including but not limitedto the ionic polarity of the protein surface. In some embodiments PHI-4polypeptides are provided having amino acid substitutions at hydrophilicresidues such as Asp, Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp, Asn, Gin,and Cys. In some embodiments the PHI-4 polypeptides are provided havingamino acid substitutions changing a Lysine or Arginine to a Glutamine,Glutamic Acid, Asparagine or Glutamic Acid; changing a Glutamic Acid orAspartic Acid to a Lysine, Asparagine or Glutamine; and changing aGlutamine to a Asparagine or Lysine.

In some embodiments PHI-4 polypeptides are provided having amino acidsubstitutions at residues in a membrane insertion loop. In someembodiments PHI-4 polypeptides are provided having amino acidsubstitutions in a membrane insertion loop between about amino acid atposition 92 (Val) and 101 (Ala) and/or at position 211 (Gly) and 220(Glu) relative to SEQ ID NO: 35.

In some embodiments PHI-4 polypeptides are provided having amino acidsubstitutions at residues and receptor binding loops. In someembodiments PHI-4 polypeptides are provided having amino acidsubstitutions at residues and receptor binding loops between about aminoacid 332 (Asp) and 340 (Asp), 395 (Asp) and 403 (Asp), 458 (Asp) and 466(Asp) relative to SEQ ID NO: 35.

In some embodiments PHI-4 polypeptides are provided having amino acidsubstitutions at residues in a protease sensitive region. In someembodiments PHI-4 polypeptides are provided having amino acidsubstitutions at residues in a protease sensitive region from aboutamino acid residues between 305 (Lys) and 316 (Lys) and 500 (Arg) and535 (Lys) relative to SEQ ID NO: 35.

By variants is intended proteins or polypeptides having an amino acidsequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the parental amino acid sequence. Insome embodiments a PHI-4 polypeptide has at least about 60%, 65%, about70%, 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greateridentity across the entire length of the amino acid sequence of SEQ IDNO: 2 or SEQ ID NOs: 51-819. In some embodiments a PHI-4 polypeptide hasat least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater identity acrossthe entire length of the amino acid sequence of SEQ ID NO: 2 or SEQ IDNOs: 51-819.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequencehaving at least 80% identity, to the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819, wherein thepolypeptide has insecticidal activity.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequencehaving at least 90% identity to the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819, wherein thepolypeptide has insecticidal activity.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequencehaving at least 95% identity to the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819 wherein the polypeptidehas insecticidal activity.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequencehaving at least 97% identity to the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819, wherein thepolypeptide has insecticidal activity.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequenceof SEQ ID NO: 4, wherein Xaa at position 9 is Gin, Lys or Glu; Xaa atposition 14 is Pro or Ala; Xaa at position 16 is Val or Asp; Xaa atposition 19 is Met or Leu; Xaa at position 22 is Gly or Ser; Xaa atposition 24 is Asp, Asn or Gin; Xaa at position 36 is Leu or Met; Xaa atposition 42 is Asp, Asn or Gin; Xaa at position 43 is Phe or Glu; Xaa atposition 46 is Glu, Asp, Asn or Gly; Xaa at position 50 is Ile or Val;Xaa at position 51 is Glu or Gin; Xaa at position 55 is Arg or Lys; Xaaat position 56 is Ser or Thr; Xaa at position 57 is Tyr or Phe; Xaa atposition 58 is Thr or Ser; Xaa at position 61 is Arg, Lys or Glu; Xaa atposition 73 is Phe or Tyr; Xaa at position 74 is Lys, Glu, Gly, Arg,Met, Leu, His or Asp; Xaa at position 76 is Asp or Gin; Xaa at position79 is Lys or Glu; Xaa at position 80 is Glu or Ser; Xaa at position 82is Glu, Ile, Leu, Tyr or Gin; Xaa at position 83 is Glu or Gin; Xaa atposition 84 is Tyr or Phe; Xaa at position 86 is Glu or Gin; Xaa atposition 87 is Lys or Gin; Xaa at position 88 is Met, Ile or Leu; Xaa atposition 90 is Gin or Glu; Xaa at position 94 is Val or Ile; Xaa atposition 97 is Arg, Asn, Asp, Glu, Gin, Gly or Ser; Xaa at position 98is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, Ile, Met, Phe, Cys,Val or Asn; Xaa at position 103 is Ala or Gly; Xaa at position 105 isLeu or Ile; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn,Glu, Cys, Ala or Arg; Xaa at position 112 is Thr or Ser; Xaa at position113 is Asp, Glu or Met; Xaa at position 117 is Thr or Ser; Xaa atposition 121 is Tyr or Phe; Xaa at position 127 is Ala or Thr; Xaa atposition 142 is Arg or Glu; Xaa at position 146 is Arg or Gin; Xaa atposition 147 is Arg, Glu or Gin; Xaa at position 148 is Asp, Phe, Pro,Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gin or Gly; Xaa at position 149is Phe or Val; Xaa at position 150 is Arg, Gin or Glu; Xaa at position151 is Asp, Ser, Ala, Asn, Trp, Val, Gin, Cys, Met, Leu, Arg or Glu; Xaaat position 153 is Leu or lie; Xaa at position 154 is Asn or Asp; Xaa atposition 155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa atposition 162 is Glu, Asp or Gin; Xaa at position 165 is Lys, Glu, Gin,Pro, Thr, Ala, Leu, Gly, Asp, Val, His, lie, Met, Trp, Phe, Tyr or Arg;Xaa at position 166 is Arg or Gin; Xaa at position 167 is Tyr, Trp orCys; Xaa at position 170 is Tyr or His; Xaa at position 171 is Tyr orPhe; Xaa at position 172 is lie, Leu or Val; Xaa at position 173 is Seror Ala; Xaa at position 174 is Glu or Gin; Xaa at position 182 is Asp orGin; Xaa at position 183 is Tyr or Val; Xaa at position 184 is Ser orThr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is Thr, Lysor lie; Xaa at position 191 is Lys or Gin; Xaa at position 193 is Asp orAsn; Xaa at position 196 is Gin, Lys, Asn, Asp, Glu, Ala, lie or Arg;Xaa at position 202 is Ala or Val; Xaa at position 203 is Glu, Thr orHis; Xaa at position 204 is Met or Ala; Xaa at position 206 is Tyr orPhe; Xaa at position 207 is Lys or Gin; Xaa at position 209 is Leu orPro; Xaa at position 210 is Val or lie; Xaa at position 214 is Lys, Seror Gin; Xaa at position 216 is Glu, Gin, Phe, Val, Tyr or Arg; Xaa atposition 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, Gin, Arg, Trp, Met,Ala, Phe, lie, Leu, Cys or Asn; Xaa at position 229 is Arg or Glu; Xaaat position 230 is Ser or Glu; Xaa at position 231 is Asn or Ser; Xaa atposition 236 is Leu or Pro; Xaa at position 245 is Met or Leu; Xaa atposition 247 is Asp or Tyr; Xaa at position 256 is Gin, Lys or Glu; Xaaat position 257 is Gin, lie, Glu, Cys, Ser, His, Trp or Met; Xaa atposition 261 is Gin, Glu or Lys; Xaa at position 264 is Glu or Gin; Xaaat position 268 is Asp or Asn; Xaa at position 276 is Ser or Ala; Xaa atposition 278 is Glu, Asn or Gin; Xaa at position 281 is Gin, Lys or Glu;Xaa at position 282 is Pro or Gly; Xaa at position 284 is Trp or Arg;Xaa at position 287 is Ala or Cys; Xaa at position 289 is Lys, Leu, Val,Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn,Cys, Gly or Ala; Xaa at position 291 is Glu or Gin; Xaa at position 292is Arg or Gin; Xaa at position 293 is Arg, Glu or Gin; Xaa at position294 is Val or Ala; Xaa at position 296 is Leu or lie; Xaa at position297 is Glu or Gin; Xaa at position 298 is Asp or Gin; Xaa at position300 is Phe or Tyr; Xaa at position 302 is Glu or Gin; Xaa at position303 is Phe or Tyr; Xaa at position 305 is Lys or Gin; Xaa at position306 is Gin or Lys; Xaa at position 309 is Gin, Lys or Glu; Xaa atposition 313 is Lys, Gin or Arg; Xaa at position 316 is Lys or Gin; Xaaat position 328 is Lys, Glu or Gin; Xaa at position 331 is Glu, Asn orGin; Xaa at position 333 is Ser, Arg, Gly, Lys, Val, Asn, Ala, His, Gin,Thr, Asp, lie, Leu, Cys or Glu; Xaa at position 334 is Gly, Arg, Lys,lie or Trp; Xaa at position 335 is Ser or Ala; Xaa at position 336 isGly or Ala; Xaa at position 337 is Ala, Val or Gly; Xaa at position 338is Ser, His, Val, Lys, Ala, Gly, Thr, lie, Glu, Met, Arg, Pro, Asp, Asnor Leu; Xaa at position 339 is Glu, Asn, Gin, lie, Pro, Met, Ser, Ala,Cys, Phe, Val, Leu, Asp, Trp, His or Arg; Xaa at position 341 is Leu orVal; Xaa at position 342 is Ala, Ser or Val; Xaa at position 343 is Valor Ile; Xaa at position 344 is Phe or Trp; Xaa at position 345 is Asn orHis; Xaa at position 346 is Pro or Ala; Xaa at position 350 is Asn orSer; Xaa at position 351 is Gly or Val; Xaa at position 354 is Met orLeu; Xaa at position 355 is Val, Ile or Leu; Xaa at position 359 is Glyor Ala; Xaa at position 362 is Asn or Ser; Xaa at position 364 is Ala orSer; Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is Pheor Ile; Xaa at position 375 is Lys or Arg; Xaa at position 380 is Leu orGly; Xaa at position 382 is Val, Asp or Leu; Xaa at position 383 is Leu,Ile or Val; Xaa at position 384 is Lys, Ala or Gly; Xaa at position 385is Ala or Gly; Xaa at position 389 is Trp or Tyr; Xaa at position 391 isArg, Leu, Glu, Gin or Asp; Xaa at position 395 is Asp or Cys; Xaa atposition 396 is Ala, Leu, Lys, Asn, Gly, Ile, Met, Arg, Tyr, Gin or His;Xaa at position 397 is Gly, Arg or Ala; Xaa at position 398 is Ser, Ginor Cys; Xaa at position 401 is Ser, His, Pro, Gly, Lys, Val, Arg, Ile,Asn, Phe, Thr, Ala, Asp, Met, Gin or Glu; Xaa at position 402 is Lys,Phe, His, Arg, Trp, Gly, Asn, Leu, Tyr, Thr, Val, Met, Pro or Ala; Xaaat position 403 is Asp, Tyr, Trp, Phe or Glu; Xaa at position 405 is Alaor Ser; Xaa at position 409 is Ala or Pro; Xaa at position 410 is Ile orVal; Xaa at position 411 is Pro or Ala; Xaa at position 412 is Pro orAla; Xaa at position 416 is Arg, Glu or Gin; Xaa at position 417 is Ala,Ser or Cys; Xaa at position 418 is Leu or Met; Xaa at position 422 isMet or Val; Xaa at position 426 is Thr or Ser; Xaa at position 436 isAsp or Lys; Xaa at position 437 is Tyr or Val; Xaa at position 438 isVal or Arg; Xaa at position 440 is Val or Leu; Xaa at position 442 isGin, Lys or Glu; Xaa at position 445 is Cys, Leu or Thr; Xaa at position447 is Asp, Lys, Tyr, Ser, Glu, Ile, Gly, Pro, Leu, Phe, Trp or Thr; Xaaat position 448 is Val or Ala; Xaa at position 449 is Gin or Glu; Xaa atposition 452 is Gin, Lys or Glu; Xaa at position 453 is Asn or Asp; Xaaat position 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, Trp, Gin,Gly, His, Asp, Leu, Thr, Pro or Asn; Xaa at position 455 is Val or Ile;Xaa at position 457 is Trp or Asn; Xaa at position 459 is Lys, Met, Val,Trp, Gin, Ile, Thr, Ser, His, Cys, Tyr, Pro, Asn, Ala, Arg or Glu; Xaaat position 460 is Gly or Ala; Xaa at position 461 is Thr or Ser; Xaa atposition 462 is Gly or Ala; Xaa at position 463 is Ala, Ser or Gly; Xaaat position 464 is Arg, Gly, His, Gin, Thr or Phe; Xaa at position 465is Lys, Asn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu;Xaa at position 466 is Asp or Arg; Xaa at position 471 is Gin, Lys orGlu; Xaa at position 497 is Asp or Gin; Xaa at position 499 is Glu orGin; Xaa at position 500 is Arg, Gin or Lys; Xaa at position 502 is Arg,Glu or Gin; Xaa at position 509 is Lys, Gin or Glu; Xaa at position 517is Gin, Cys, Asn, Val or Pro; Xaa at position 518 is Glu or Gin; Xaa atposition 520 is Lys, Gin or Glu; Xaa at position 525 is Gin or Lys; andXaa at position 527 is Gin, Lys, Pro, Cys, Glu, Ser, His, Phe or Trp;and having one or more amino acid substitutions at positions designatedas Xaa in SEQ ID NO: 4 and wherein the PHI-4 polypeptide has increasedinsecticidal activity compared to SEQ ID NO: 2; and amino acidsubstitutions, deletions, insertions, and fragments thereof, andcombinations thereof.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequenceof SEQ ID NO: 4 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, inany combination, at residues designated by Xaa in SEQ ID NO: 4 comparedto the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequenceof SEQ ID NO: 4 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 amino acid substitutions, in any combination, atresidues designated by Xaa in SEQ ID NO: 4 compared to the native aminoacid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequencehaving at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identityto the amino acid sequence of SEQ ID NO: 4.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequenceof SEQ ID NO: 3, wherein Xaa at position 24 is Asp or Asn; Xaa atposition 42 is Asp or Asn; Xaa at position 43 is Phe or Glu; Xaa atposition 46 is Glu or Asn; Xaa at position 74 is Lys, Glu or Gly; Xaa atposition 79 is Lys or Glu; Xaa at position 82 is Glu, Ile, Leu or Tyr;Xaa at position 97 is Arg, Asn, Asp, Glu, Gin or Gly; Xaa at position 98is Tyr or Phe; Xaa at position 99 is Lys, Leu, Tyr, Ile or Met; Xaa atposition 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa at position 147is Arg or Glu; Xaa at position 148 is Asp, Phe or Pro; Xaa at position150 is Arg or Gin; Xaa at position 151 is Asp, Ser, Ala or Asn; Xaa atposition 153 is Leu or Ile; Xaa at position 162 is Glu or Gin; Xaa atposition 165 is Lys, Glu or Gin; Xaa at position 166 is Arg or Gin; Xaaat position 171 is Tyr or Phe; Xaa at position 174 is Glu or Gin; Xaa atposition 182 is Asp or Gin; Xaa at position 196 is Gin, Lys, Asn or Asp;Xaa at position 203 is Glu, Thr or His; Xaa at position 206 is Tyr orPhe; Xaa at position 216 is Glu or Gin; Xaa at position 220 is Glu, His,Asp, Thr, Tyr, Val, Ser or Gin; Xaa at position 247 is Asp or Tyr; Xaaat position 256 is Gin or Lys; Xaa at position 257 is Gin or Ile; Xaa atposition 261 is Gin or Glu; Xaa at position 278 is Glu or Asn; Xaa atposition 281 is Gin, Lys or Glu; Xaa at position 289 is Lys, Leu, Val,Pro, Glu, Gin, Tyr, Thr or Asp; Xaa at position 293 is Arg, Glu or Gin;Xaa at position 313 is Lys or Gin; Xaa at position 328 is Lys, Glu orGin; Xaa at position 333 is Ser, Gly, Lys, Val or Asn; Xaa at position334 is Gly, Arg, Lys or Ile; Xaa at position 336 is Gly or Ala; Xaa atposition 338 is Ser, His, Val, Lys or Ala; Xaa at position 339 is Glu,Asn, Ile or Pro; Xaa at position 343 is Val or Ile; Xaa at position 346is Pro or Ala; Xaa at position 355 is Val or Ile; Xaa at position 359 isGly or Ala; Xaa at position 391 is Arg, Glu or Gin; Xaa at position 396is Ala, Leu, Lys, Asn or Gly; Xaa at position 401 is Ser, His, Pro, Gly,Lys, Val or Arg; Xaa at position 402 is Lys, Phe, His, Arg, Gly, Trp,Thr, Asn, Tyr or Met; Xaa at position 403 is Asp or Tyr; Xaa at position411 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position416 is Arg or Glu; Xaa at position 417 is Ala or Ser; Xaa at position418 is Leu or Met; Xaa at position 426 is Thr or Ser; Xaa at position440 is Val or Leu; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu orIle; Xaa at position 452 is Gin, Lys or Glu; Xaa at position 454 is Arg,Tyr, Met, Ser, Val, Ile, Lys, Phe, Trp or Gin; Xaa at position 455 isVal or Ile; Xaa at position 459 is Lys, Met, Val, Trp, Gin, Ile or Tyr;Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly or Ala;Xaa at position 463 is Ala or Ser; Xaa at position 464 is Arg, Gly orHis; Xaa at position 465 is Lys, Asn, Val, Met, Pro, Gly or Arg; Xaa atposition 471 is Gin, Lys or Glu; Xaa at position 500 is Arg or Gin; Xaaat position 509 is Lys or Gin; Xaa at position 520 is Lys, Gin or Glu;and Xaa at position 527 is Gin, Lys, Pro, Cys or Glu; and having one ormore amino acid substitutions at positions designated as Xaa in SEQ IDNO: 3 and wherein the PHI-4 polypeptide has increased insecticidalactivity compared to SEQ ID NO: 35; and amino acid substitutions,deletions, insertions, and fragments thereof, and combinations thereof.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequenceof SEQ ID NO: 3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75 or 76 amino acid substitutions, in anycombination, at residues designated by Xaa in SEQ ID NO: 3 compared tothe native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments PHI-4 polypeptide comprises an amino acid sequenceof SEQ ID NO: 3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53 or 54 amino acid substitutions, in any combination, at residuesdesignated by Xaa in SEQ ID NO: 3 compared to the native amino acid atthe corresponding position of SEQ ID NO: 2.

In some embodiments PHI-4 polypeptide comprises an amino acid sequenceof SEQ ID NO: 3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14 or15 amino acid substitutions, in any combination, at residues designatedby Xaa in SEQ ID NO: 3 compared to the native amino acid at thecorresponding position of SEQ ID NO: 2.

In some embodiments a PHI-4 polypeptide comprises an amino acid sequencehaving at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identityto the amino acid sequence of SEQ ID NO: 3.

In some embodiments a PHI-4 polypeptide comprises one or more amino acidsubstitutions compared to the native amino acid at position 40, 42, 43,46, 52, 97, 98, 99, 145, 150, 151, 153, 163, 171, 172, 182, 196, 206,210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342,346, 354, 355, 370, 389, 393, 396, 401, 402, 403, 410, 412, 416, 417,426, 442, 447, 452, 454, 455, 457, 461, 462, 500, 509, 520 or 527 of SEQID NO: 35.

In some embodiments a PHI-4 polypeptide comprises one or more amino acidsubstitutions compared to the native amino acid at position 40, 42, 43,46, 52, 97, 98, 99, 145, 150, 151, 153, 163, 171, 172, 182, 196, 206,210, 216, 220, 278, 283, 289, 293, 328, 333, 334, 336, 338, 339, 342,346, 354, 355, 370, 389, 393, 396, 401, 402, 403, 410, 412, 416, 417,426, 442, 447, 452, 454, 455, 457, 461, 462, 500, 509, 520 or 527 of SEQID NO: 35 wherein the amino acid at position 40 is Leu or Ile; the aminoacid at position 42 is Asp or Asn; the amino acid at position 43 is Pheor Glu; the amino acid at position 46 is Glu or Asn; the amino acid atposition 52 is Ile or Val; the amino acid at position 97 is Arg, Asp,Glu or Asn; the amino acid at position 98 is Tyr or Phe; the amino acidat position 99 is Lys or Leu; the amino acid at position 145 is Leu orVal; the amino acid at position 150 is Arg or Gin; the amino acid atposition 151 is Asp or Ser; the amino acid at position 153 is Leu orIle; the amino acid at position 163 is Leu or Val; the amino acid atposition 171 is Tyr or Phe; the amino acid at position 172 is Ile orLeu; the amino acid at position 182 is Asp or Gin; the amino acid atposition 196 is Gin or Asn; the amino acid at position 206 is Tyr orPhe; the amino acid at position 210 is Val or Ile; the amino acid atposition 216 is Glu or Gin; the amino acid at position 220 is Glu, Gin,His or Asp; the amino acid at position 278 is Glu or Asn; the amino acidat position 283 is Ile or Val; the amino acid at position 289 is Lys,Gin or Leu; the amino acid at position 293 is Arg, Gin or Glu; the aminoacid at position 328 is Lys or Glu; the amino acid at position 333 isSer, Lys or Val; the amino acid at position 334 is Gly, Lys or Arg; theamino acid at position 336 is Gly or Ala; the amino acid at position 338is Ser or Val; the amino acid at position 339 is Glu, Asn or Gin; theamino acid at position 342 is Ala or Ser; the amino acid at position 346is Pro or Ala; the amino acid at position 354 is Met or Leu; the aminoacid at position 355 is Val or Ile; the amino acid at position 370 isHis or Arg; the amino acid at position 389 is Trp or Leu; the amino acidat position 393 is Trp or Leu; the amino acid at position 396 is Ala,Leu, Lys, Thr or Gly; the amino acid at position 401 is Ser, His, Gly,Lys or Pro; the amino acid at position 402 is Lys, His, Gly or Trp; theamino acid at position 403 is Asp or Tyr; the amino acid at position 410is Ile or Val; the amino acid at position 412 is Pro or Ala; the aminoacid at position 416 is Arg or Glu; the amino acid at position 417 isAla or Ser; the amino acid at position 426 is Thr or Ser; the amino acidat position 442 is Gin or Glu; the amino acid at position 447 is Asp orLys; the amino acid at position 452 is Gin or Lys; the amino acid atposition 454 is Arg or Gin; the amino acid at position 455 is Val orIle; the amino acid at position 457 is Trp or Asn; the amino acid atposition 461 is Thr or Ser; the amino acid at position 462 is Gly orAla; the amino acid at position 500 is Arg or Gin; the amino acid atposition 509 is Lys or Gin; the amino acid at position 520 is Lys, Gluor Gin; and the amino acid at position 527 is Gin or Lys., and aminoacid deletions, insertions and fragments thereof, and combinationsthereof.

In some embodiments the PHI-4 polypeptide comprising one or more aminoacid substitutions at position 86, 359, 399, 464, 465, 466, 467, 468,499 or 517.

In some embodiments the PHI-4 polypeptide comprising one or more aminoacid substitutions at position 86, 359, 399, 464, 465, 466, 467, 468,499 or 517, wherein the amino acid at position 86 is Glu or Thr; theamino acid at position 359 is Gly or Ala; the amino acid at position 399is Gly or Ala; the amino acid at position 464 is Arg, Ala, Lys, Asp orAsn; the amino acid at position 465 is Lys or Met, the amino acid atposition 467 is Val, Ala, Leu or Thr; the amino acid at position 468 isSer or Leu; the amino acid at position 499 is Glu or Ala, or the aminoacid at position 517 is Glu or Arg.

In some embodiments exemplary PHI-4 polypeptides are encoded by thepolynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ IDNO: 11, SEQ ID NOS: 24-30.

In some embodiments a PHI-4 polypeptide includes variants where an aminoacid that is part of a proteolytic cleavage site is changed to anotheramino acid to eliminate or alter the proteolytic cleavage at that site.In some embodiments the proteolytic cleavage is by a protease in theinsect gut. In other embodiments the proteolytic cleavage is by a plantprotease in the transgenic plant.

In some embodiments exemplary PHI-4 polypeptides are the polypeptidesshown in Table 3, Table 4, Table 5, Table 6, Table 7, Megatable 1, andMegatable 2 and combinations of the amino substitutions thereof as wellas amino acid deletions, and or insertions and fragments thereof.

In some embodiments a PHI-4 polypeptide is encoded by a nucleic acidmolecule that hybridizes under stringent conditions to the nucleic acidmolecule of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NOS:24-30. Variants include polypeptides that differ in amino acid sequencedue to mutagenesis. Variant proteins encompassed by the disclosure arebiologically active, that is they continue to possess the desiredbiological activity (i.e. pesticidal activity) of the native protein. By“retains activity” is intended that the variant will have at least about30%, at least about 50%, at least about 70% or at least about 80% of theinsecticidal activity of the native protein. In some embodiments, thevariants may have improved activity over the native protein.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present disclosure andmay be used in the methods of the present disclosure. It will beunderstood that, when expressed in plants, it will be necessary to alterthe alternate start codon to ATG for proper translation.

In another aspect the PHI-4 polypeptide may be expressed as a precursorprotein with an intervening sequence that catalyzes multi-step, posttranslational protein splicing. Protein splicing involves the excisionof an intervening sequence from a polypeptide with the concomitantjoining of the flanking sequences to yield a new polypeptide (Chong, etal., (1996) J. Biol. Chem. 271:22159-22168). This intervening sequenceor protein splicing element, referred to as inteins, which catalyzetheir own excision through three coordinated reactions at the N-terminaland C-terminal splice junctions: an acyl rearrangement of the N-terminalcysteine or Serine; a transesterfication reaction between the twotermini to form a branched ester or thioester intermediate and peptidebond cleavage coupled to cyclization of the intein C-terminal asparagineto free the intein (Evans, et al., (2000) J. Biol. Chem. 275:9091-9094.The elucidation of the mechanism of protein splicing has led to a numberof intein-based applications (Comb, et al., U.S. Pat. No. 5,496,714;Comb, et al., U.S. Pat. No. 5,834,247; Camarero and Muir, (1999) J.Amer. Chem. Soc. 121:5597-5598; Chong, et al., (1997) Gene 192:271-281,Chong, et al., (1998) Nucleic Acids Res. 26:5109-5115; Chong, et al.,(1998) J. Biol. Chem. 273:10567-10577; Cotton, et al., (1999) J. Am.Chem. Soc. 121:1100-1101; Evans, et al., (1999) J. Biol. Chem.274:18359-18363; Evans, et al., (1999) J. Biol. Chem. 274:3923-3926;Evans, et al., (1998) Protein Sci. 7:2256-2264; Evans, et al., (2000) J.Biol. Chem. 275:9091-9094; Iwai and Pluckthun, (1999) FEBS Lett.459:166-172; Mathys, et al., (1999) Gene 231:1-13; Mills, et al., (1998)Proc. Natl. Acad. Sci. USA 95:3543-3548; Muir, et al., (1998) Proc.Natl. Acad. Sci. USA 95:6705-6710; Otomo, et al., (1999) Biochemistry38:16040-16044; Otomo, et al., (1999) J. Biolmol. NMR 14:105-114; Scott,et al., (1999) Proc. Natl. Acad. Sci. USA 96:13638-13643; Severinov andMuir, (1998) J. Biol. Chem. 273:16205-16209; Shingledecker, et al.,(1998) Gene 207:187-195; Southworth, et al., (1998) EMBO J. 17:918-926;Southworth, et al., (1999) Biotechniques 27:110-120; Wood, et al.,(1999) Nat. Biotechnol. 17:889-892; Wu, et al., (1998a) Proc. Natl.Acad. Sci. USA 95:9226-9231; Wu, et al., (1998b) Biochim Biophys Acta1387:422-432; Xu, et al., (1999) Proc. Natl. Acad. Sci. USA 96:388-393;Yamazaki, et al., (1998) J. Am. Chem. Soc. 120:5591-5592). For theapplication of inteins in plant transgenes see Yang, J, et al.,(Transgene Res 15:583-593 (2006)) and Evans, et al., (Annu. Rev. PlantBiol. 56:375-392, (2005)).

In another aspect the PHI-4 polypeptide may be encoded by two separategenes where the intein of the precursor protein comes from the twogenes, referred to as a split-intein and the two portions of theprecursor are joined by a peptide bond formation. This peptide bondformation is accomplished by intein-mediated trans-splicing. For thispurpose, a first and a second expression cassette comprising the twoseparate genes further code for inteins capable of mediating proteintrans-splicing. By trans-splicing, the proteins and polypeptides encodedby the first and second fragments may be linked by peptide bondformation. Trans-splicing inteins may be selected from the nucleolar andorganellar genomes of different organisms including eukaryotes,archaebacteria and eubacteria. Inteins that may be used for are listedat neb.com/neb/inteins.html, which can be accessed on the world-wide webusing the “www” prefix). The nucleotide sequence coding for an inteinmay be split into a 5′ and a 3′ part that code for the 5′ and the 3′part of the intein, respectively. Sequence portions not necessary forintein splicing (e.g., homing endonuclease domain) may be deleted. Theintein coding sequence is split such that the 5′ and the 3′ parts arecapable of trans-splicing. For selecting a suitable splitting site ofthe intein coding sequence, the considerations published by Southworth,et al., (1998) EMBO J. 17:918-926 may be followed. In constructing thefirst and the second expression cassette, the 5′ intein coding sequenceis linked to the 3′ end of the first fragment coding for the N-terminalpart of the PHI-4 polypeptide and the 3′ intein coding sequence islinked to the 5′ end of the second fragment coding for the C-terminalpart of the PHI-4 polypeptide.

In general, the trans-splicing partners can be designed using any splitintein, including any naturally-occurring or artificially-split splitintein. Several naturally-occurring split inteins are known, forexample: the split intein of the DnaE gene of Synechocystis sp. PCC6803(see, Wu, et al., (1998) Proc Natl Acad Sci USA 95(16):9226-31 andEvans, et al., (2000) J Biol Chem 275(13):9091-4 and of the DnaE genefrom Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett580(7):1853-8). Non-split inteins have been artificially split in thelaboratory to create new split inteins, for example: the artificiallysplit Ssp DnaB intein (see, Wu, et al., (1998) Biochim Biophys Acta1387:422-32) and split Sce VMA intein (see, Brenzel, et al., (2006)Biochemistry 45(6):1571-8) and an artificially split fungal mini-intein(see, Elleuche, et al., (2007) Biochem Biophys Res Commun 355(3):830-4).There are also intein databases available that catalogue known inteins(see, for example the online-database available at:bioinformatics.weizmann.ac.il\pietro/inteins/Inteinstable.html, whichcan be accessed on the world-wide web using the “www” prefix).

Naturally-occurring non-split inteins may have endonuclease or otherenzymatic activities that can typically be removed when designing anartificially-split split intein. Such mini-inteins or minimized splitinteins are well known in the art and are typically less than 200 aminoacid residues long (see, Wu, et al., (1998) Biochim Biophys Acta1387:422-32). Suitable split inteins may have other purificationenabling polypeptide elements added to their structure, provided thatsuch elements do not inhibit the splicing of the split intein or areadded in a manner that allows them to be removed prior to splicing.Protein splicing has been reported using proteins that comprisebacterial intein-like (BIL) domains (see, Amitai, et al., (2003) MolMicrobiol 47:61-73) and hedgehog (Hog) auto-processing domains (thelatter is combined with inteins when referred to as the Hog/inteinsuperfamily or HINT family (see, Dassa, et al., (2004) J Biol Chem. 27932001-7) and domains such as these may also be used to prepareartificially-split inteins. In particular, non-splicing members of suchfamilies may be modified by molecular biology methodologies to introduceor restore splicing activity in such related species. Recent studiesdemonstrate that splicing can be observed when a N-terminal split inteincomponent is allowed to react with a C-terminal split intein componentnot found in nature to be its “partner”; for example, splicing has beenobserved utilizing partners that have as little as 30 to 50% homologywith the “natural” splicing partner (see, Dassa, et al., (2007)Biochemistry 46(1):322-30). Other such mixtures of disparate splitintein partners have been shown to be unreactive one with another (see,Brenzel, et al., 2006 Biochemistry 45(6):1571-8). However, it is withinthe ability of a person skilled in the relevant art to determine whethera particular pair of polypeptides is able to associate with each otherto provide a functional intein, using routine methods and without theexercise of inventive skill.

In another aspect the PHI-4 polypeptide is a circular permuted variant.In certain embodiments the PHI-4 polypeptide is a circular permutedvariant of the polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4or SEQ ID NOs: 51-819. The development of recombinant DNA methods hasmade it possible to study the effects of sequence transposition onprotein folding, structure and function. The approach used in creatingnew sequences resembles that of naturally occurring pairs of proteinsthat are related by linear reorganization of their amino acid sequences(Cunningham, et al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222;Teather and Erfle, (1990) J. Bacteriol. 172:3837-3841; Schimming, etal., (1992) Eur. J. Biochem. 204:13-19; Yamiuchi and Minamikawa, (1991)FEBS Lett. 260:127-130; MacGregor, et al., (1996) FEBS Lett.378:263-266). The first in vitro application of this type ofrearrangement to proteins was described by Goldenberg and Creighton (J.Mol. Biol. 165:407-413, 1983). In creating a circular permuted variant anew N-terminus is selected at an internal site (breakpoint) of theoriginal sequence, the new sequence having the same order of amino acidsas the original from the breakpoint until it reaches an amino acid thatis at or near the original C-terminus. At this point the new sequence isjoined, either directly or through an additional portion of sequence(linker), to an amino acid that is at or near the original N-terminusand the new sequence continues with the same sequence as the originaluntil it reaches a point that is at or near the amino acid that wasN-terminal to the breakpoint site of the original sequence, this residueforming the new C-terminus of the chain. The length of the amino acidsequence of the linker can be selected empirically or with guidance fromstructural information, or by using a combination of the two approaches.When no structural information is available, a small series of linkerscan be prepared for testing using a design whose length is varied inorder to span a range from 0 to 50 Å and whose sequence is chosen inorder to be consistent with surface exposure (hydrophilicity, Hopp andWoods, (1983) Mol. Immunol. 20:483-489; Kyte and Doolittle, (1982) J.Mol. Biol. 157:105-132; solvent exposed surface area, Lee and Richards,(1971) J. Mol. Biol. 55:379-400) and the ability to adopt the necessaryconformation without deranging the configuration of the pesticidalpolypeptide (conformationally flexible; Karplus and Schulz, (1985)Naturwissenschaften 72:212-213. Assuming an average of translation of2.0 to 3.8 Å per residue, this would mean the length to test would bebetween 0 to 30 residues, with 0 to 15 residues being the preferredrange. Exemplary of such an empirical series would be to constructlinkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated ntimes, where n is 1, 2, 3 or 4. Those skilled in the art will recognizethat there are many such sequences that vary in length or compositionthat can serve as linkers with the primary consideration being that theybe neither excessively long nor short (cf., Sandhu, (1992) Critical Rev.Biotech. 12:437-462); if they are too long, entropy effects will likelydestabilize the three-dimensional fold, and may also make foldingkinetically impractical, and if they are too short, they will likelydestabilize the molecule because of torsional or steric strain. Thoseskilled in the analysis of protein structural information will recognizethat using the distance between the chain ends, defined as the distancebetween the c-alpha carbons, can be used to define the length of thesequence to be used, or at least to limit the number of possibilitiesthat must be tested in an empirical selection of linkers. They will alsorecognize that it is sometimes the case that the positions of the endsof the polypeptide chain are ill-defined in structural models derivedfrom x-ray diffraction or nuclear magnetic resonance spectroscopy data,and that when true, this situation will therefore need to be taken intoaccount in order to properly estimate the length of the linker required.From those residues whose positions are well defined are selected tworesidues that are close in sequence to the chain ends, and the distancebetween their c-alpha carbons is used to calculate an approximate lengthfor a linker between them. Using the calculated length as a guide,linkers with a range of number of residues (calculated using 2 to 3.8 Åper residue) are then selected. These linkers may be composed of theoriginal sequence, shortened or lengthened as necessary, and whenlengthened the additional residues may be chosen to be flexible andhydrophilic as described above; or optionally the original sequence maybe substituted for using a series of linkers, one example being theGly-Gly-Gly-Ser cassette approach mentioned above; or optionally acombination of the original sequence and new sequence having theappropriate total length may be used. Sequences of pesticidalpolypeptides capable of folding to biologically active states can beprepared by appropriate selection of the beginning (amino terminus) andending (carboxyl terminus) positions from within the originalpolypeptide chain while using the linker sequence as described above.Amino and carboxyl termini are selected from within a common stretch ofsequence, referred to as a breakpoint region, using the guidelinesdescribed below. A novel amino acid sequence is thus generated byselecting amino and carboxyl termini from within the same breakpointregion. In many cases the selection of the new termini will be such thatthe original position of the carboxyl terminus immediately preceded thatof the amino terminus. However, those skilled in the art will recognizethat selections of termini anywhere within the region may function, andthat these will effectively lead to either amino acid deletions oradditions to the amino or carboxyl portions of the new sequence. It is acentral tenet of molecular biology that the primary amino acid sequenceof a protein dictates folding to the three-dimensional structurenecessary for expression of its biological function. Methods are knownto those skilled in the art to obtain and interpret three-dimensionalstructural information using x-ray diffraction of single proteincrystals or nuclear magnetic resonance spectroscopy of proteinsolutions. Examples of structural information that are relevant to theidentification of breakpoint regions include the location and type ofprotein secondary structure (alpha and 3-10 helices, parallel andanti-parallel beta sheets, chain reversals and turns, and loops; Kabschand Sander, (1983) Biopolymers 22:2577-2637; the degree of solventexposure of amino acid residues, the extent and type of interactions ofresidues with one another (Chothia, (1984) Ann. Rev. Biochem.53:537-572) and the static and dynamic distribution of conformationsalong the polypeptide chain (Alber and Mathews, (1987) Methods Enzymol.154:511-533). In some cases additional information is known aboutsolvent exposure of residues; one example is a site ofpost-translational attachment of carbohydrate which is necessarily onthe surface of the protein. When experimental structural information isnot available, or is not feasible to obtain, methods are also availableto analyze the primary amino acid sequence in order to make predictionsof protein tertiary and secondary structure, solvent accessibility andthe occurrence of turns and loops. Biochemical methods are alsosometimes applicable for empirically determining surface exposure whendirect structural methods are not feasible; for example, using theidentification of sites of chain scission following limited proteolysisin order to infer surface exposure (Gentile and Salvatore, (1993) Eur.J. Biochem. 218:603-621). Thus using either the experimentally derivedstructural information or predictive methods (e.g., Srinivisan and Rose,(1995) Proteins: Struct., Funct. & Genetics 22:81-99) the parental aminoacid sequence is inspected to classify regions according to whether ornot they are integral to the maintenance of secondary and tertiarystructure. The occurrence of sequences within regions that are known tobe involved in periodic secondary structure (alpha and 3-10 helices,parallel and anti-parallel beta sheets) are regions that should beavoided. Similarly, regions of amino acid sequence that are observed orpredicted to have a low degree of solvent exposure are more likely to bepart of the so-called hydrophobic core of the protein and should also beavoided for selection of amino and carboxyl termini. In contrast, thoseregions that are known or predicted to be in surface turns or loops, andespecially those regions that are known not to be required forbiological activity, are the preferred sites for location of theextremes of the polypeptide chain. Continuous stretches of amino acidsequence that are preferred based on the above criteria are referred toas a breakpoint region. Polynucleotides encoding circular permuted PHI-4polypeptides with new N-terminus/C-terminus which contain a linkerregion separating the original C-terminus and N-terminus can be madeessentially following the method described in Mullins, et al., (1994) J.Am. Chem. Soc. 116:5529-5533. Multiple steps of polymerase chainreaction (PCR) amplifications are used to rearrange the DNA sequenceencoding the primary amino acid sequence of the protein. Polynucleotidesencoding circular permuted PHI-4 polypeptides with newN-terminus/C-terminus which contain a linker region separating theoriginal C-terminus and N-terminus can be made based on thetandem-duplication method described in Horlick, et al., (1992) ProteinEng. 5:427-431. Polymerase chain reaction (PCR) amplification of the newN-terminus/C-terminus genes is performed using a tandemly duplicatedtemplate DNA.

In another aspect fusion proteins are provided that include within itsamino acid sequence an amino acid sequence comprising a PHI-4polypeptide including but not limited to the polypeptide of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819 and active fragmentsthereof.

In another aspect fusion proteins are provided comprising a PHI-4polypeptide and a second pesticidal polypeptide such a Cry protein.Methods for design and construction of fusion proteins (andpolynucleotides encoding same) are known to those of skill in the art.Polynucleotides encoding a PHI-4 polypeptide may be fused to signalsequences which will direct the localization of the PHI-4 polypeptide toparticular compartments of a prokaryotic or eukaryotic cell and/ordirect the secretion of the PHI-4 polypeptide of the embodiments from aprokaryotic or eukaryotic cell. For example, in E. coli, one may wish todirect the expression of the protein to the periplasmic space. Examplesof signal sequences or proteins (or fragments thereof) to which thePHI-4 polypeptide may be fused in order to direct the expression of thepolypeptide to the periplasmic space of bacteria include, but are notlimited to, the pelB signal sequence, the maltose binding protein (MBP)signal sequence, MBP, the ompA signal sequence, the signal sequence ofthe periplasmic E. coli heat-labile enterotoxin B-subunit, and thesignal sequence of alkaline phosphatase. Several vectors arecommercially available for the construction of fusion proteins whichwill direct the localization of a protein, such as the pMAL series ofvectors (particularly the pMAL-p series) available from New EnglandBiolabs. In a specific embodiment, the PHI-4 polypeptide may be fused tothe pelB pectate lyase signal sequence to increase the efficiency ofexpression and purification of such polypeptides in Gram-negativebacteria (see, U.S. Pat. Nos. 5,576,195 and 5,846,818). Plant plastidtransit peptide/polypeptide fusions are well known in the art (see, U.S.Pat. No. 7,193,133). Apoplast transit peptides such as rice or barleyalpha-amylase secretion signal are also well known in the art. Theplastid transit peptide is generally fused N-terminal to the polypeptideto be targeted (e.g., the fusion partner). In one embodiment, the fusionprotein consists essentially of the peptide transit plastid and thePHI-4 polypeptide to be targeted. In another embodiment, the fusionprotein comprises the peptide transit plastid and the polypeptide to betargeted. In such embodiments, the plastid transit peptide is preferablyat the N-terminus of the fusion protein. However, additional amino acidresidues may be N-terminal to the plastid transit peptide providing thatthe fusion protein is at least partially targeted to a plastid. In aspecific embodiment, the plastid transit peptide is in the N-terminalhalf, N-terminal third or N-terminal quarter of the fusion protein. Mostor all of the plastid transit peptide is generally cleaved from thefusion protein upon insertion into the plastid. The position of cleavagemay vary slightly between plant species, at different plantdevelopmental stages, as a result of specific intercellular conditionsor the particular combination of transit peptide/fusion partner used. Inone embodiment, the plastid transit peptide cleavage is homogenous suchthat the cleavage site is identical in a population of fusion proteins.In another embodiment, the plastid transit peptide is not homogenous,such that the cleavage site varies by 1-10 amino acids in a populationof fusion proteins. The plastid transit peptide can be recombinantlyfused to a second protein in one of several ways. For example, arestriction endonuclease recognition site can be introduced into thenucleotide sequence of the transit peptide at a position correspondingto its C-terminal end and the same or a compatible site can beengineered into the nucleotide sequence of the protein to be targeted atits N-terminal end. Care must be taken in designing these sites toensure that the coding sequences of the transit peptide and the secondprotein are kept “in frame” to allow the synthesis of the desired fusionprotein. In some cases, it may be preferable to remove the initiatormethionine codon of the second protein when the new restriction site isintroduced. The introduction of restriction endonuclease recognitionsites on both parent molecules and their subsequent joining throughrecombinant DNA techniques may result in the addition of one or moreextra amino acids between the transit peptide and the second protein.This generally does not affect targeting activity as long as the transitpeptide cleavage site remains accessible and the function of the secondprotein is not altered by the addition of these extra amino acids at itsN-terminus. Alternatively, one skilled in the art can create a precisecleavage site between the transit peptide and the second protein (withor without its initiator methionine) using gene synthesis (Stemmer, etal., (1995) Gene 164:49-53) or similar methods. In addition, the transitpeptide fusion can intentionally include amino acids downstream of thecleavage site. The amino acids at the N-terminus of the mature proteincan affect the ability of the transit peptide to target proteins toplastids and/or the efficiency of cleavage following protein import.This may be dependent on the protein to be targeted. See, e.g., Comai,et al., (1988) J. Biol. Chem. 263(29):15104-9.

In some embodiments fusion proteins are provide comprising a PHI-4polypeptide, a pesticidal protein such as a cry protein, and an aminoacid linker.

In some embodiments fusion proteins are provided represented by aformula selected from the group consisting of

R¹-L-R²,R²-L-R¹,R¹—R² or R²—R¹

where R¹ is a PHI-4 polypeptide, R² is a pesticidal protein with adifferent but complementary activity to the PHI-4 polypeptide, includingbut not limited to cry proteins; a polypeptide that increases thesolubility and/or stability of the PHI-4 polypeptide; or a transitpeptide or leader sequence. The R¹ polypeptide is fused either directlyor through a linker segment to the R² polypeptide. The term “directly”defines fusions in which the polypeptides are joined without a peptidelinker. Thus L represents a chemical bound or polypeptide segment towhich both R¹ and R² are fused in frame, most commonly L is a linearpeptide to which R¹ and R² are bound by amide bonds linking the carboxyterminus of R¹ to the amino terminus of L and carboxy terminus of L tothe amino terminus of R². By “fused in frame” is meant that there is notranslation termination or disruption between the reading frames of R¹and R². The linking group (L) is generally a polypeptide of between 1and 500 amino acids in length. The linkers joining the two molecules arepreferably designed to (1) allow the two molecules to fold and actindependently of each other, (2) not have a propensity for developing anordered secondary structure which could interfere with the functionaldomains of the two proteins, (3) have minimal hydrophobic or chargedcharacteristic which could interact with the functional protein domainsand (4) provide steric separation of R¹ and R² such that R¹ and R² couldinteract simultaneously with their corresponding receptors on a singlecell. Typically surface amino acids in flexible protein regions includeGly, Asn and Ser. Virtually any permutation of amino acid sequencescontaining Gly, Asn and Ser would be expected to satisfy the abovecriteria for a linker sequence. Other neutral amino acids, such as Thrand Ala, may also be used in the linker sequence. Additional amino acidsmay also be included in the linkers due to the addition of uniquerestriction sites in the linker sequence to facilitate construction ofthe fusions.

In some embodiments the linkers comprise sequences selected from thegroup of formulas: (Gly₃Ser)_(n), (Gly₄Ser)_(n), (Gly₅Ser)_(n),(Gly_(n)Ser)_(n) or (AlaGlySer)_(n) where n is an integer. One exampleof a highly-flexible linker is the (GlySer)-rich spacer region presentwithin the pill protein of the filamentous bacteriophages, e.g.,bacteriophages M13 or fd (Schaller, et al., 1975). This region providesa long, flexible spacer region between two domains of the pill surfaceprotein. Also included are linkers in which an endopeptidase recognitionsequence is included. Such a cleavage site may be valuable to separatethe individual components of the fusion to determine if they areproperly folded and active in vitro. Examples of various endopeptidasesinclude, but are not limited to, Plasmin, Enterokinase, Kallikerin,Urokinase, Tissue Plasminogen activator, clostripain, Chymosin,Collagenase, Russell's Viper Venom Protease, Postproline cleavageenzyme, V8 protease, Thrombin and factor Xa. In some embodiments thelinker comprises the amino acids EEKKN from the multi-gene expressionvehicle (MGEV), which is cleaved by vacuolar proteases as disclosed inUS 2007/0277263. In other embodiments, peptide linker segments from thehinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgEprovide an angular relationship between the attached polypeptides.Especially useful are those hinge regions where the cysteines arereplaced with serines. Preferred linkers of the present inventioninclude sequences derived from murine IgG gamma 2b hinge region in whichthe cysteines have been changed to serines. The fusion proteins are notlimited by the form, size or number of linker sequences employed and theonly requirement of the linker is that functionally it does notinterfere adversely with the folding and function of the individualmolecules of the fusion.

In another aspect chimeric PHI-4 polypeptide are provided that arecreated through joining two or more portions of genes, which originallyencoded separate insecticidal proteins from different species, to createa chimeric gene. The translation of the chimeric gene results in asingle chimeric pesticidal polypeptide with regions, motifs or domainsderived from each of the original polypeptides. In certain embodimentsthe chimeric protein comprises portions, motifs, or domains of PHI-4polypeptides in any combination. In certain embodiments the chimericinsecticidal polypeptide includes but not limited to the polypeptides ofSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819.

It is recognized that DNA sequences may be altered by various methods,and that these alterations may result in DNA sequences encoding proteinswith amino acid sequences different than that encoded by the wild-type(or native) pesticidal protein. These proteins may be altered in variousways including amino acid substitutions, amino acid deletions, aminoacid truncations, and insertions of one or more amino acids, includingup to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155 or more amino acid substitutions, amino acid deletionsand/or insertions or combinations thereof compared to SEQ ID NO: 35 SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819. In someembodiments a PHI-4 polypeptide comprises the deletion of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28 or more amino acids from the C-terminus of the PHI-4polypeptide relative to the amino acid position of SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4 or SEQ ID NOs: 51-819. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of a PHI-4 polypeptide can be prepared by mutations inthe DNA. This may also be accomplished by one of several forms ofmutagenesis and/or in directed evolution. In some aspects, the changesencoded in the amino acid sequence will not substantially affect thefunction of the protein. Such variants will possess the desiredpesticidal activity. However, it is understood that the ability of aPHI-4 polypeptide to confer pesticidal activity may be improved by theuse of such techniques upon the compositions of this disclosure.

For example, conservative amino acid substitutions may be made at one ormore, predicted, nonessential amino acid residues. A “nonessential”amino acid residue is a residue that can be altered from the wild-typesequence of a PHI-4 polypeptide without altering the biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include: amino acidswith basic side chains (e.g., lysine, arginine, histidine); acidic sidechains (e.g., aspartic acid, glutamic acid); polar, negatively chargedresidues and their amides (e.g., aspartic acid, asparagine, glutamic,acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine); small aliphatic,nonpolar or slightly polar residues (e.g., Alanine, serine, threonine,proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); largealiphatic, nonpolar residues (e.g., methionine, leucine, isoleucine,valine, cystine); beta-branched side chains (e.g., threonine, valine,isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine); large aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues, or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the embodiments (e.g., residues that areidentical in an alignment of homologous proteins). Examples of residuesthat are conserved but that may allow conservative amino acidsubstitutions and still retain activity include, for example, residuesthat have only conservative substitutions between all proteins containedin an alignment of similar or related toxins to the sequences of theembodiments (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment homologous proteins).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues. Guidance as to appropriate amino acid substitutionsthat do not affect biological activity of the protein of interest may befound in the model of Dayhoff, et al., (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, (1982) J Mol Biol.157(1):105-32). It is accepted that the relative hydropathic characterof the amino acid contributes to the secondary structure of theresultant protein, which in turn defines the interaction of the proteinwith other molecules, for example, enzymes, substrates, receptors, DNA,antibodies, antigens and the like.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, ibid). These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9) and arginine(−4.5). In making such changes, the substitution of amino acids whosehydropathic indices are within +2 is preferred, those which are within+1 are particularly preferred and those within +0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include amino acid insertions, amino aciddeletions, or amino acid alterations introduced by modern molecularmethods, such as PCR, including PCR amplifications that alter or extendthe protein coding sequence by virtue of inclusion of amino acidencoding sequences in the oligonucleotides utilized in the PCRamplification. Alternatively, the protein sequences added can includeentire protein-coding sequences, such as those used commonly in the artto generate protein fusions. Such fusion proteins are often used to (1)increase expression of a protein of interest (2) introduce a bindingdomain, enzymatic activity, or epitope to facilitate either proteinpurification, protein detection, or other experimental uses known in theart (3) target secretion or translation of a protein to a subcellularorganelle, such as the periplasmic space of Gram-negative bacteria,mitochondria or chloroplasts of plants or the endoplasmic reticulum ofeukaryotic cells, the latter of which often results in glycosylation ofthe protein.

In specific embodiments, the substitution is an alanine for the nativeamino acid at the recited position(s). Also encompassed are the nucleicacid sequence(s) encoding the variant protein or polypeptide.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentPHI-4 polypeptide coding regions can be used to create a new PHI-4polypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneand other known pesticidal genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedinsecticidal activity. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al.,(1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri, et al., (1998) Nature 391:288-291 and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredPHI-4 polypeptides. Domains may be swapped between PHI-4 polypeptides,resulting in hybrid or chimeric toxins with improved pesticidal activityor target spectrum. Methods for generating recombinant proteins andtesting them for pesticidal activity are well known in the art (see, forexample, Naimov, et al., (2001) Appl. Environ. Microbiol. 67:5328-5330;de Maagd, et al., (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, etal., (1991) J. Biol. Chem. 266:17954-17958; Schnepf, et al., (1990) J.Biol. Chem. 265:20923-20930; Rang, et al., 91999) Appl. Environ.Microbiol. 65:2918-2925).

Antibodies

Antibodies to a PHI-4 polypeptide of the embodiments, or to variants orfragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane,(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

A kit for detecting the presence of a PHI-4 polypeptide, or detectingthe presence of a nucleotide sequence encoding a PHI-4 polypeptide, in asample is provided. In one embodiment, the kit provides antibody-basedreagents for detecting the presence of a PHI-4 polypeptide in a tissuesample. In another embodiment, the kit provides labeled nucleic acidprobes useful for detecting the presence of one or more polynucleotidesencoding PHI-4 polypeptide(s). The kit is provided along withappropriate reagents and controls for carrying out a detection method,as well as instructions for use of the kit.”

Receptor Identification and Isolation

Receptors to the PHI-4 polypeptide of the embodiments, or to variants orfragments thereof, are also encompassed. Methods for identifyingreceptors are well known in the art (see, Hofmann, et. al., (1988) Eur.J. Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. 27277-27282)can be employed to identify and isolate the receptor that recognizes thePHI-4 polypeptides using the brush-border membrane vesicles fromsusceptible insects. In addition to the radioactive labeling methodlisted in the cited literatures, PHI-4 polypeptide can be labeled withfluorescent dye and other common labels such as streptavidin.Brush-border membrane vesicles (BBMV) of susceptible insects such assoybean looper and stink bugs can be prepared according to the protocolslisted in the references and separated on SDS-PAGE gel and blotted onsuitable membrane. Labeled PHI-4 polypeptides can be incubated withblotted membrane of BBMV and labeled the PHI-4 polypeptides can beidentified with the labeled reporters. Identification of protein band(s)that interact with the PHI-4 polypeptides can be detected by N-terminalamino acid gas phase sequencing or mass spectrometry based proteinidentification method (Patterson, (1998) 10(22):1-24, Current Protocolin Molecular Biology published by John Wiley & Son Inc). Once theprotein is identified, the corresponding gene can be cloned from genomicDNA or cDNA library of the susceptible insects and binding affinity canbe measured directly with the PHI-4 polypeptides. Receptor function forinsecticidal activity by the PHI-4 polypeptides can be verified byaccomplished by RNAi type of gene knock out method (Rajagopal, et al.,(2002) J. Biol. Chem. 277:46849-46851).

Nucleotide Constructs, Expression Cassettes and Vectors

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructsparticularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules and sequences. Further, thenucleotide constructs, nucleotide molecules and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant and insect cells, bacteria, yeast,baculovirus, protozoa, nematodes and algae. The transformed organismcomprises a DNA molecule of the embodiments, an expression cassettecomprising the DNA molecule or a vector comprising the expressioncassette, which may be stably incorporated into the genome of thetransformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand where necessary to join two protein coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the PHI-4 polypeptide gene sequence to be under thetranscriptional regulation of the regulatory regions. The DNA constructmay additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

In some embodiments the DNA construct may also include a transcriptionalenhancer sequence. As used herein, the term an “enhancer” refers to aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Various enhancers areknown in the art including for example, introns with gene expressionenhancing properties in plants (US Patent Application Publication Number2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1(see, for example, NCBI sequence S94464)), the omega enhancer or theomega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNAed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990)EMBO J. 9:1685-96) and the enhancers of U.S. Pat. No. 7,803,992 may alsobe used, each of which is incorporated by reference. The above list oftranscriptional enhancers is not meant to be limiting. Any appropriatetranscriptional enhancer can be used in the embodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. For example, although nucleic acid sequencesof the embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons as these preferences have been shown todiffer (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, themaize-preferred codon for a particular amino acid may be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants is listed in Table 4 of Murray, et al., supra. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, and 5,436,391 and Murray, et al.,(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli, oreukaryotic cells such as yeast, insect, amphibian or mammalian cells ormonocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak,et al., (1991) Nature 353:90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al.,(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie,et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York),pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, etal., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987)Plant Physiol. 84:965-968. Such constructs may also contain a “signalsequence” or “leader sequence” to facilitate co-translational orpost-translational transport of the peptide to certain intracellularstructures such as the chloroplast (or other plastid), endoplasmicreticulum, or Golgi apparatus.

By “signal sequence” is intended a sequence that is known or suspectedto result in cotranslational or post-translational peptide transportacross the cell membrane. In eukaryotes, this typically involvessecretion into the Golgi apparatus, with some resulting glycosylation.Insecticidal toxins of bacteria are often synthesized as protoxins,which are protolytically activated in the gut of the target pest (Chang,(1987) Methods Enzymol. 153:507-516). In some embodiments, the signalsequence is located in the native sequence, or may be derived from asequence of the embodiments. By “leader sequence” is intended anysequence that when translated, results in an amino acid sequencesufficient to trigger co-translational transport of the peptide chain toa subcellular organelle. Thus, this includes leader sequences targetingtransport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria and the like. Nuclear-encoded proteins targeted to thechloroplast thylakoid lumen compartment have a characteristic bipartitetransit peptide, composed of a stromal targeting signal peptide and alumen targeting signal peptide. The stromal targeting information is inthe amino-proximal portion of the transit peptide. The lumen targetingsignal peptide is in the carboxyl-proximal portion of the transitpeptide, and contains all the information for targeting to the lumen.Recent research in proteomics of the higher plant chloroplast hasachieved in the identification of numerous nuclear-encoded lumenproteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al.Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta1503:350-356, 2001), the lumen targeting signal peptide of which canpotentially be used in accordance with the present invention. About 80proteins from Arabidopsis, as well as homologous proteins from spinachand garden pea, are reported by Kieselbach et al., PhotosynthesisResearch, 78:249-264, 2003. In particular, table 2 of this publication,which is incorporated into the description herewith by reference,discloses 85 proteins from the chloroplast lumen, identified by theiraccession number (see also US Patent Application Publication2009/09044298). In addition, the recently published draft version of therice genome (Goff et al, Science 296:92-100, 2002) is a suitable sourcefor lumen targeting signal peptide which may be used in accordance withthe present invention.

Suitable chloroplast transit peptides (CTP) are well known to oneskilled in the art also include chimeric CTPs comprising but not limitedto, an N-terminal domain, a central domain or a C-terminal domain from aCTP from Oryza sativa 1-deoxy-D xyulose-5-Phosphate Synthase, Oryzasativa-Superoxide dismutase, Oryza sativa-soluble starch synthase, Oryzasativa-NADP-dependent Malic acid enzyme, Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2, Oryzasativa-L-Ascorbate peroxidase 5, Oryza sativa-Phosphoglucan waterdikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malatedehydrogenase, Zea Mays Thioredoxin M-type US Patent ApplicationPublication 2012/0304336).

The PHI-4 polypeptide gene to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible, orother promoters for expression in the host organism. Suitableconstitutive promoters for use in a plant host cell include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; thecore CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); riceactin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin(Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound-inducible promoters. Such wound-inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath.28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford, et al.,(1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992)Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol.22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene(Corderok, et al., (1994) Plant J. 6(2):141-150) and the like, hereinincorporated by reference.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. PlantPathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4:645-656 and VanLoon, (1985) Plant Mol. Virol. 4:111-116. See also, WO 1999/43819,herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau, et al., (1987) PlantMol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 andYang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen,et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl.Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201;Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible) and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1α promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced PHI-4polypeptide expression within a particular plant tissue.Tissue-preferred promoters include those discussed in Yamamoto, et al.,(1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant CellPhysiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet.254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168;Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, etal., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996)Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant CellPhysiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ.20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred or root-specific promoters are known and can be selectedfrom the many available from the literature or isolated de novo fromvarious compatible species. See, for example, Hire, et al., (1992) PlantMol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetasegene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061(root-specific control element in the GRP 1.8 gene of French bean);Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-lengthcDNA clone encoding cytosolic glutamine synthetase (GS), which isexpressed in roots and root nodules of soybean). See also, Bogusz, etal., (1990) Plant Cell 2(7):633-641, where two root-specific promotersisolated from hemoglobin genes from the nitrogen-fixing nonlegumeParasponia andersonii and the related non-nitrogen-fixing nonlegumeTrema tomentosa are described. The promoters of these genes were linkedto a 3-glucuronidase reporter gene and introduced into both thenonlegume Nicotiana tabacum and the legume Lotus corniculatus, and inboth instances root-specific promoter activity was preserved. Leach andAoyagi, (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes(see, Plant Science (Limerick) 79(1):69-76). They concluded thatenhancer and tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri, et al., (1989) used gene fusion to lacZ to show thatthe Agrobacterium T-DNA gene encoding octopine synthase is especiallyactive in the epidermis of the root tip and that the TR2′ gene is rootspecific in the intact plant and stimulated by wounding in leaf tissue,an especially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1′gene fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol.29(4):759-772) and roIB promoter (Capana, et al., (1994) Plant Mol.Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase) (see, U.S. Pat. No. 6,225,529,herein incorporated by reference). Gamma-zein and Glb-1 areendosperm-specific promoters. For dicots, seed-specific promotersinclude, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3)(Jofuku, K. D. and Goldberg, R. B. Plant Cell 1:1079-1093, 1989), beanβ-phaseolin, napin, 3-conglycinin, glycinin 1, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also, WO2000/12733, where seed-preferred promoters from end1 and end2 genes aredisclosed; herein incorporated by reference. In dicots, seed specificpromoters include but are not limited to seed coat promoter fromArabidopsis, pBAN; and the early seed promoters from Arabidopsis, p26,p63, and p63tr (U.S. Pat. Nos. 7,294,760 and 7,847,153). A promoter thathas “preferred” expression in a particular tissue is expressed in thattissue to a greater degree than in at least one other plant tissue. Sometissue-preferred promoters show expression almost exclusively in theparticular tissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of about 1/1000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts is intended. Alternatively,it is recognized that the term “weak promoters” also encompassespromoters that drive expression in only a few cells and not in others togive a total low level of expression. Where a promoter drives expressionat unacceptably high levels, portions of the promoter sequence can bedeleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142 and 6,177,611, herein incorporated by reference.

The above list of promoters is not meant to be limiting. Any appropriatepromoter can be used in the embodiments.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton,(1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992)Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, etaL,(1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566;Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen,(1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993)Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol.Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad.Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill,et al., (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

Plant Transformation

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide or polypeptides gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide or polypeptides into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. By “plant” isintended whole plants, plant organs (e.g., leaves, stems, roots, etc.),seeds, plant cells, propagules, embryos and progeny of the same. Plantcells can be differentiated or undifferentiated (e.g. callus, suspensionculture cells, protoplasts, leaf cells, root cells, phloem cells, andpollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Led transformation (WO 2000/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research9:71-78. Additional transformation procedures can be found inWeissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the PHI-4 polypeptide or variants and fragmentsthereof directly into the plant or the introduction of the PHI-4polypeptide transcript into the plant. Such methods include, forexample, microinjection or particle bombardment. See, for example,Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al.,(1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad.Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.Alternatively, the PHI-4 polypeptide polynucleotide can be transientlytransformed into the plant using techniques known in the art. Suchtechniques include viral vector system and the precipitation of thepolynucleotide in a manner that precludes subsequent release of the DNA.Thus, transcription from the particle-bound DNA can occur, but thefrequency with which it is released to become integrated into the genomeis greatly reduced. Such methods include the use of particles coatedwith polyethylimine (PEI; Sigma #P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853, all of which are herein incorporated by reference.Briefly, the polynucleotide of the embodiments can be contained intransfer cassette flanked by two non-identical recombination sites. Thetransfer cassette is introduced into a plant have stably incorporatedinto its genome a target site which is flanked by two non-identicalrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

Plant transformation vectors may be comprised of one or more DNA vectorsneeded for achieving plant transformation. For example, it is a commonpractice in the art to utilize plant transformation vectors that arecomprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors”. Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the pesticidal gene are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux, (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g., immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g., Hiei, et al., (1994) ThePlant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park, (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strainsand the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that therecombinant proteins of the embodiments may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired PHI-4 polypeptide. It is alsorecognized that such a viral polyprotein, comprising at least a portionof the amino acid sequence of a PHI-4 polypeptide of the embodiments,may have the desired pesticidal activity. Such viral polyproteins andthe nucleotide sequences that encode for them are encompassed by theembodiments. Methods for providing plants with nucleotide constructs andproducing the encoded proteins in the plants, which involve viral DNA orRNA molecules are known in the art. See, for example, U.S. Pat. Nos.5,889,191; 5,889,190; 5,866,785; 5,589,367 and 5,316,931, hereinincorporated by reference.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The methodrelies on particle gun delivery of DNA containing a selectable markerand targeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride, et al.,(1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves, and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta) and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewing's fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithi); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrumsecundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum);carpet grass (Axonopus affinis); centipede grass (Eremochloaophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum(Paspalum vaginatum); blue gramma (Bouteloua gracilis); buffalo grass(Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, favabean, lentils, chickpea, etc.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell, (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, (2001) supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled 32P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, (2001) supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, (2001) supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, (2001) supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thePHI-4 polypeptide.

Stacking of Traits in Transgenic Plant

Transgenic plants may comprise a stack of one or more insecticidalpolynucleotides disclosed herein with one or more additionalpolynucleotides resulting in the production or suppression of multiplepolypeptide sequences. Transgenic plants comprising stacks ofpolynucleotide sequences can be obtained by either or both oftraditional breeding methods or through genetic engineering methods.These methods include, but are not limited to, breeding individual lineseach comprising a polynucleotide of interest, transforming a transgenicplant comprising a gene disclosed herein with a subsequent gene, andco-transformation of genes into a single plant cell. As used herein, theterm “stacked” includes having the multiple traits present in the sameplant (i.e., both traits are incorporated into the nuclear genome, onetrait is incorporated into the nuclear genome and one trait isincorporated into the genome of a plastid, or both traits areincorporated into the genome of a plastid). In one non-limiting example,“stacked traits” comprise a molecular stack where the sequences arephysically adjacent to each other. A trait, as used herein, refers tothe phenotype derived from a particular sequence or groups of sequences.Co-transformation of genes can be carried out using singletransformation vectors comprising multiple genes or genes carriedseparately on multiple vectors. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO1999/25855 and WO 1999/25853, all of which are herein incorporated byreference.

In some embodiments the polynucleotides encoding the PHI-4 polypeptidesdisclosed herein, alone or stacked with one or more additional insectresistance traits can be stacked with one or more additional inputtraits (e.g., herbicide resistance, fungal resistance, virus resistanceor stress tolerance, disease resistance, male sterility, stalk strength,and the like) or output traits (e.g., increased yield, modifiedstarches, improved oil profile, balanced amino acids, high lysine ormethionine, increased digestibility, improved fiber quality, droughtresistance, and the like). Thus, the polynucleotide embodiments can beused to provide a complete agronomic package of improved crop qualitywith the ability to flexibly and cost effectively control any number ofagronomic pests.

Transgenes useful for stacking include but are not limited to:1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., (1994) Science266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae), McDowell and Woffenden, (2003)Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) TransgenicRes. 11(6):567-82. A plant resistant to a disease is one that is moreresistant to a pathogen as compared to the wild type plant.

(B) Genes encoding a Bacillus thuringiensis protein, a derivativethereof or a synthetic polypeptide modeled thereon. See, for example,Geiser, et al., (1986) Gene 48:109, who disclose the cloning andnucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNAmolecules encoding delta-endotoxin genes can be purchased from AmericanType Culture Collection (Rockville, Md.), for example, under ATCCAccession Numbers 40098, 67136, 31995 and 31998. Other non-limitingexamples of Bacillus thuringiensis transgenes being geneticallyengineered are given in the following patents and patent applicationsand hereby are incorporated by reference for this purpose: U.S. Pat.Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594,6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826,7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556,7,329,736, 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862,7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846,7,858,849 and WO 1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581and WO 1997/40162.

(C) A polynucleotide encoding an insect-specific hormone or pheromonesuch as an ecdysteroid and juvenile hormone, a variant thereof, amimetic based thereon or an antagonist or agonist thereof. See, forexample, the disclosure by Hammock, et al., (1990) Nature 344:458, ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone.

(D) A polynucleotide encoding an insect-specific peptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of, Regan, (1994) J. Biol. Chem. 269:9 (expressioncloning yields DNA coding for insect diuretic hormone receptor); Pratt,et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin isidentified in Diploptera puntata); Chattopadhyay, et al., (2004)Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J NatProd 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 andVasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also, U.S.Pat. No. 5,266,317 to Tomalski, et al., who disclose genes encodinginsect-specific toxins.

(E) A polynucleotide encoding an enzyme responsible for ahyperaccumulation of a monoterpene, a sesquiterpene, a steroid,hydroxamic acid, a phenylpropanoid derivative or another non-proteinmolecule with insecticidal activity.

(F) A polynucleotide encoding an enzyme involved in the modification,including the post-translational modification, of a biologically activemolecule; for example, a glycolytic enzyme, a proteolytic enzyme, alipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, ahydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, anelastase, a chitinase and a glucanase, whether natural or synthetic.See, PCT Application WO 1993/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Numbers 39637 and 67152. See also, Kramer, etal., (1993) Insect Biochem. Molec. Biol. 23:691, who teach thenucleotide sequence of a cDNA encoding tobacco hookworm chitinase andKawalleck, et al., (1993) Plant Molec. Biol. 21:673, who provide thenucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and U.S.Pat. Nos. 6,563,020; 7,145,060 and 7,087,810.

(G) A polynucleotide encoding a molecule that stimulates signaltransduction. For example, see the disclosure by Botella, et al., (1994)Plant Molec. Biol. 24:757, of nucleotide sequences for mung beancalmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol.104:1467, who provide the nucleotide sequence of a maize calmodulin cDNAclone.

(H) A polynucleotide encoding a hydrophobic moment peptide. See, PCTApplication WO 1995/16776 and U.S. Pat. No. 5,580,852 disclosure ofpeptide derivatives of Tachyplesin which inhibit fungal plant pathogens)and PCT Application WO 1995/18855 and U.S. Pat. No. 5,607,914 (teachessynthetic antimicrobial peptides that confer disease resistance).

(I) A polynucleotide encoding a membrane permease, a channel former or achannel blocker. For example, see the disclosure by Jaynes, et al.,(1993) Plant Sci. 89:43, of heterologous expression of a cecropin-betalytic peptide analog to render transgenic tobacco plants resistant toPseudomonas solanacearum.

(J) A gene encoding a viral-invasive protein or a complex toxin derivedtherefrom. For example, the accumulation of viral coat proteins intransformed plant cells imparts resistance to viral infection and/ordisease development effected by the virus from which the coat proteingene is derived, as well as by related viruses. See, Beachy, et al.,(1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistancehas been conferred upon transformed plants against alfalfa mosaic virus,cucumber mosaic virus, tobacco streak virus, potato virus X, potatovirus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaicvirus. Id.

(K) A gene encoding an insect-specific antibody or an immunotoxinderived therefrom. Thus, an antibody targeted to a critical metabolicfunction in the insect gut would inactivate an affected enzyme, killingthe insect. Cf. Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUMON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

(L) A gene encoding a virus-specific antibody. See, for example,Tavladoraki, et al., (1993) Nature 366:469, who show that transgenicplants expressing recombinant antibody genes are protected from virusattack.

(M) A polynucleotide encoding a developmental-arrestive protein producedin nature by a pathogen or a parasite. Thus, fungal endoalpha-1,4-D-polygalacturonases facilitate fungal colonization and plantnutrient release by solubilizing plant cell wallhomo-alpha-1,4-D-galacturonase. See, Lamb, et al., (1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart, etal., (1992) Plant J. 2:367.

(N) A polynucleotide encoding a developmental-arrestive protein producedin nature by a plant. For example, Logemann, et al., (1992)Bio/Technology 10:305, have shown that transgenic plants expressing thebarley ribosome-inactivating gene have an increased resistance to fungaldisease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, (1995) Current Biology5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich, (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, (1993) PI. Physiol.101:709-712 and Parijs, et al., (1991) Planta 183:258-264 and Bushnell,et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S.patent application Ser. Nos. 09/950,933; 11/619,645; 11/657,710;11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. LysMReceptor-like kinases for the perception of chitin fragments as a firststep in plant defense response against fungal pathogens (US2012/0110696).

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see, U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) A polynucleotide encoding a Cystatin and cysteine proteinaseinhibitors. See, U.S. Pat. No. 7,205,453.

(S) Defensin genes. See, WO 2003/000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See, e.g., PCT ApplicationWO 1996/30517; PCT Application WO 1993/19181, WO 2003/033651 and Urwin,et al., (1998) Planta 204:472-479, Williamson, (1999) Curr Opin PlantBio. 2(4):327-31; U.S. Pat. Nos. 6,284,948 and 7,301,069 and miR164genes (WO 2012/058266).

(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker, et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent Application Publication US 2009/0035765 and incorporated byreference for this purpose. This includes the Rcg locus that may beutilized as a single locus conversion.

2. Transgenes that Confer Resistance to a Herbicide, for Example:

(A) A polynucleotide encoding resistance to a herbicide that inhibitsthe growing point or meristem, such as an imidazolinone or asulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449,respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870;5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and5,378,824; U.S. patent application Ser. No. 11/683,737 and InternationalPublication WO 1996/33270.

(B) A polynucleotide encoding a protein for resistance to Glyphosate(resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase(EPSP) and aroA genes, respectively) and other phosphono compounds suchas glufosinate (phosphinothricin acetyl transferase (PAT) andStreptomyces hygroscopicus phosphinothricin acetyl transferase (bar)genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No.4,940,835 to Shah, et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes.See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497;5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;5,094,945, 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667;4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and5,491,288 and International Publications EP 1173580; WO 2001/66704; EP1173581 and EP 1173582, which are incorporated herein by reference forthis purpose. Glyphosate resistance is also imparted to plants thatexpress a gene encoding a glyphosate oxidoreductase enzyme as describedmore fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which areincorporated herein by reference for this purpose. In additionglyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.Pat. Nos. 7,462,481; 7,405,074 and US Patent Application PublicationNumber US 2008/0234130. A DNA molecule encoding a mutant aroA gene canbe obtained under ATCC Accession Number 39256, and the nucleotidesequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 toComai. EP Application Number 0 333 033 to Kumada, et al., and U.S. Pat.No. 4,975,374 to Goodman, et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EP ApplicationNumbers 0 242 246 and 0 242 236 to Leemans, et al., De Greef, et al.,(1989) Bio/Technology 7:61, describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. See also, U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616 B1 and 5,879,903, which are incorporated herein by referencefor this purpose. Exemplary genes conferring resistance to phenoxyproprionic acids and cyclohexones, such as sethoxydim and haloxyfop, arethe Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall, et al.,(1992) Theor. Appl. Genet. 83:435.

(C) A polynucleotide encoding a protein for resistance to herbicide thatinhibits photosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell3:169, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA moleculescontaining these genes are available under ATCC Accession Numbers 53435,67441 and 67442. Cloning and expression of DNA coding for a glutathioneS-transferase is described by Hayes, et al., (1992) Biochem. J. 285:173.

(D) A polynucleotide encoding a protein for resistance to Acetohydroxyacid synthase, which has been found to make plants that express thisenzyme resistant to multiple types of herbicides, has been introducedinto a variety of plants (see, e.g., Hattori, et al., (1995) Mol GenGenet. 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994)Plant Physiol 106:17), genes for glutathione reductase and superoxidedismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genesfor various phosphotransferases (Datta, et al., (1992) Plant Mol Biol20:619).

(E) A polynucleotide encoding resistance to a herbicide targetingProtoporphyrinogen oxidase (protox) which is necessary for theproduction of chlorophyll. The protox enzyme serves as the target for avariety of herbicidal compounds. These herbicides also inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are resistant to these herbicides are described in U.S.Pat. Nos. 6,288,306 B1; 6,282,837 B1 and 5,767,373 and InternationalPublication WO 2001/12825.

(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodesthe aryloxyalkanoate dioxygenase (AAD-1) protein. The trait conferstolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate(commonly referred to as “fop” herbicides such as quizalofop)herbicides. The aad-1 gene, itself, for herbicide tolerance in plantswas first disclosed in WO 2005/107437 (see also, US 2009/0093366). Theaad-12 gene, derived from Delftia acidovorans, which encodes thearyloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides bydeactivating several herbicides with an aryloxyalkanoate moiety,including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxyauxins (e.g., fluroxypyr, triclopyr).

(G) A polynucleotide encoding a herbicide resistant dicambamonooxygenase disclosed in US Patent Application Publication2003/0135879 for imparting dicamba tolerance;

(H) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn)disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance;

(I) A polynucleotide molecule encoding phytoene (crtl) described inMisawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994)Plant J. 6:481-489 for norflurazon tolerance.

3. Transgenes that Confer or Contribute to an Altered GrainCharacteristic

Such as:

(A) Altered fatty acids, for example, by

(1) Down-regulation of stearoyl-ACP to increase stearic acid content ofthe plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA89:2624 and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn).

(2) Elevating oleic acid via FAD-2 gene modification and/or decreasinglinolenic acid via FAD-3 gene modification (see, U.S. Pat. Nos.6,063,947; 6,323,392; 6,372,965 and WO 1993/11245).

(3) Altering conjugated linolenic or linoleic acid content, such as inWO 2001/12800.

(4) Altering LEC1, AGP, Dek1, Superall, mil ps, various Ipa genes suchas Ipal, Ipa3, hpt or hggt. For example, see, WO 2002/42424, WO1998/22604, WO 2003/011015, WO 2002/057439, WO 2003/011015, U.S. Pat.Nos. 6,423,886, 6,197,561, 6,825,397 and US Patent ApplicationPublication Numbers US 2003/0079247, US 2003/0204870 and Rivera-Madrid,et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624.

(5) Genes encoding delta-8 desaturase for making long-chainpolyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 and 8,338,152),delta-9 desaturase for lowering saturated fats (U.S. Pat. No.8,063,269), Primula A6-desaturase for improving omega-3 fatty acidprofiles.

(6) Isolated nucleic acids and proteins associated with lipid and sugarmetabolism regulation, in particular, lipid metabolism protein (LMP)used in methods of producing transgenic plants and modulating levels ofseed storage compounds including lipids, fatty acids, starches or seedstorage proteins and use in methods of modulating the seed size, seednumber, seed weights, root length and leaf size of plants (EP 2404499).

(7) Altering expression of a High-Level Expression of Sugar-Inducible 2(HS12) protein in the plant to increase or decrease expression of HS12in the plant. Increasing expression of HS12 increases oil content whiledecreasing expression of HS12 decreases abscisic acid sensitivity and/orincreases drought resistance (US Patent Application Publication Number2012/0066794).

(8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oilcontent in plant seed, particular to increase the levels of omega-3fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (USPatent Application Publication Number 2011/0191904).

(9) Nucleic acid molecules encoding wrinkled1-like polypeptides formodulating sugar metabolism (U.S. Pat. No. 8,217,223).

(B) Altered phosphorus content, for example, by the

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see, Van Hartingsveldt, et al., (1993) Gene 127:87, for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) Modulating a gene that reduces phytate content. In maize, this, forexample, could be accomplished, by cloning and then re-introducing DNAassociated with one or more of the alleles, such as the LPA alleles,identified in maize mutants characterized by low levels of phytic acid,such as in WO 2005/113778 and/or by altering inositol kinase activity asin WO 2002/059324, US Patent Application Publication Number2003/0009011, WO 2003/027243, US Patent Application Publication Number2003/0079247, WO 1999/05298, U.S. Pat. Nos. 6,197,561, 6,291,224,6,391,348, WO 2002/059324, US Patent Application Publication Number2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147.

(C) Altered carbohydrates affected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or, a genealtering thioredoxin such as NTR and/or TRX (see, U.S. Pat. No.6,531,648. which is incorporated by reference for this purpose) and/or agamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, U.S.Pat. No. 6,858,778 and US Patent Application Publication Number2005/0160488, US Patent Application Publication Number 2005/0204418,which are incorporated by reference for this purpose). See, Shiroza, etal., (1988) J. Bacteriol. 170:810 (nucleotide sequence of Streptococcusmutant fructosyltransferase gene), Steinmetz, et al., (1985) Mol. Gen.Genet. 200:220 (nucleotide sequence of Bacillus subtilis levansucrasegene), Pen, et al., (1992) Bio/Technology 10:292 (production oftransgenic plants that express Bacillus licheniformis alpha-amylase),Elliot, et al., (1993) Plant Molec. Biol. 21:515 (nucleotide sequencesof tomato invertase genes), Segaard, et al., (1993) J. Biol. Chem.268:22480 (site-directed mutagenesis of barley alpha-amylase gene) andFisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starchbranching enzyme II), WO 1999/10498 (improved digestibility and/orstarch extraction through modification of UDP-D-xylose 4-epimerase,Fragile 1 and 2, Ref1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method ofproducing high oil seed by modification of starch levels (AGP)). Thefatty acid modification genes mentioned herein may also be used toaffect starch content and/or composition through the interrelationshipof the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see, U.S. Pat. No. 6,787,683,US Patent Application Publication Number 2004/0034886 and WO 2000/68393involving the manipulation of antioxidant levels and WO 2003/082899through alteration of a homogentisate geranyl geranyl transferase(hggt).

(E) Altered essential seed amino acids. For example, see, U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO 1999/40209 (alteration of amino acid compositions inseeds), WO 1999/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO 1998/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO 1998/56935 (plant amino acid biosyntheticenzymes), WO 1998/45458 (engineered seed protein having higherpercentage of essential amino acids), WO 1998/42831 (increased lysine),U.S. Pat. No. 5,633,436 (increasing sulfur amino acid content), U.S.Pat. No. 5,559,223 (synthetic storage proteins with defined structurecontaining programmable levels of essential amino acids for improvementof the nutritional value of plants), WO 1996/01905 (increasedthreonine), WO 1995/15392 (increased lysine), US Patent ApplicationPublication Number 2003/0163838, US Patent Application PublicationNumber 2003/0150014, US Patent Application Publication Number2004/0068767, U.S. Pat. No. 6,803,498, WO 2001/79516.

4. Genes that Control Male-Sterility:

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT (WO 2001/29237).

(B) Introduction of various stamen-specific promoters (WO 1992/13956, WO1992/13957).

(C) Introduction of the barnase and the barstar gene (Paul, et al.,(1992) Plant Mol. Biol. 19:611-622).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014 and 6,265,640, all of which are hereby incorporatedby reference.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see, Lyznik, et al., (2003) Plant Cell Rep 21:925-932 andWO 1999/25821, which are hereby incorporated by reference. Other systemsthat may be used include the Gin recombinase of phage Mu (Maeser, etal., (1991) Vicki Chandler, The Maize Handbook ch. 118 (Springer-Verlag1994), the Pin recombinase of E. coli (Enomoto, et al., 1983) and theR/RS system of the pSRi plasmid (Araki, et al., 1992).

6. Genes that Affect Abiotic Stress Resistance

Including but not limited to flowering, ear and seed development,enhancement of nitrogen utilization efficiency, altered nitrogenresponsiveness, drought resistance or tolerance, cold resistance ortolerance and salt resistance or tolerance and increased yield understress.

(A) For example, see: WO 2000/73475 where water use efficiency isaltered through alteration of malate; U.S. Pat. Nos. 5,892,009,5,965,705, 5,929,305, 5,891,859, 6,417,428, 6,664,446, 6,706,866,6,717,034, 6,801,104, WO 2000/060089, WO 2001/026459, WO 2001/035725, WO2001/034726, WO 2001/035727, WO 2001/036444, WO 2001/036597, WO2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO2002/079403, WO 2003/013227, WO 2003/013228, WO 2003/014327, WO2004/031349, WO 2004/076638, WO 199809521.

(B) WO 199938977 describing genes, including CBF genes and transcriptionfactors effective in mitigating the negative effects of freezing, highsalinity and drought on plants, as well as conferring other positiveeffects on plant phenotype.

(C) US Patent Application Publication Number 2004/0148654 and WO2001/36596 where abscisic acid is altered in plants resulting inimproved plant phenotype such as increased yield and/or increasedtolerance to abiotic stress.

(D) WO 2000/006341, WO 2004/090143, U.S. Pat. Nos. 7,531,723 and6,992,237 where cytokinin expression is modified resulting in plantswith increased stress tolerance, such as drought tolerance, and/orincreased yield. Also see, WO 2002/02776, WO 2003/052063, JP2002/281975, U.S. Pat. No. 6,084,153, WO 2001/64898, U.S. Pat. Nos.6,177,275 and 6,107,547 (enhancement of nitrogen utilization and alterednitrogen responsiveness).

(E) For ethylene alteration, see, US Patent Application PublicationNumber 2004/0128719, US Patent Application Publication Number2003/0166197 and WO 2000/32761.

(F) For plant transcription factors or transcriptional regulators ofabiotic stress, see, e.g., US Patent Application Publication Number2004/0098764 or US Patent Application Publication Number 2004/0078852.

(G) Genes that increase expression of vacuolar pyrophosphatase such asAVP1 (U.S. Pat. No. 8,058,515) for increased yield; nucleic acidencoding a HSFA4 or a HSFA5 (Heat Shock Factor of the class A4 or A5)polypeptides, an oligopeptide transporter protein (OPT4-like)polypeptide; a plastochron2-like (PLA2-like) polypeptide or a Wuschelrelated homeobox 1-like (WOX1-like) polypeptide (U. Patent ApplicationPublication Number US 2011/0283420).

(H) Down regulation of polynucleotides encoding poly (ADP-ribose)polymerase (PARP) proteins to modulate programmed cell death (U.S. Pat.No. 8,058,510) for increased vigor.

(I) Polynucleotide encoding DTP21 polypeptides for conferring droughtresistance (US Patent Application Publication Number US 2011/0277181).

(J) Nucleotide sequences encoding ACC Synthase 3 (ACS3) proteins formodulating development, modulating response to stress, and modulatingstress tolerance (US Patent Application Publication Number US2010/0287669).

(K) Polynucleotides that encode proteins that confer a drought tolerancephenotype (DTP) for conferring drought resistance (WO 2012/058528).

(L) Tocopherol cyclase (TC) genes for conferring drought and salttolerance (US Patent Application Publication Number 2012/0272352).

(M) CAAX amino terminal family proteins for stress tolerance (U.S. Pat.No. 8,338,661).

(N) Mutations in the SAL1 encoding gene have increased stress tolerance,including increased drought resistant (US Patent Application PublicationNumber 2010/0257633).

(O) Expression of a nucleic acid sequence encoding a polypeptideselected from the group consisting of: GRF polypeptide, RAA1-likepolypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptideincreasing yield-related traits (US Patent Application PublicationNumber 2011/0061133).

(P) Modulating expression in a plant of a nucleic acid encoding a ClassIII Trehalose Phosphate Phosphatase (TPP) polypeptide for enhancingyield-related traits in plants, particularly increasing seed yield (USPatent Application Publication Number 2010/0024067).

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g., WO1997/49811 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/14414 (CON), WO1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO 1999/49064(GI), WO 2000/46358 (FR1), WO 1997/29123, U.S. Pat. Nos. 6,794,560,6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO2004/031349 (transcription factors).

7. Genes that Confer Increased Yield

(A) A transgenic crop plant transformed by a1-AminoCyclopropane-1-Carboxylate Deaminase-like Polypeptide (ACCDP)coding nucleic acid, wherein expression of the nucleic acid sequence inthe crop plant results in the plant's increased root growth, and/orincreased yield, and/or increased tolerance to environmental stress ascompared to a wild type variety of the plant (U.S. Pat. No. 8,097,769).

(B) Over-expression of maize zinc finger protein gene (Zm-ZFP1) using aseed preferred promoter has been shown to enhance plant growth, increasekernel number and total kernel weight per plant (US Patent ApplicationPublication Number 2012/0079623).

(C) Constitutive over-expression of maize lateral organ boundaries (LOB)domain protein (Zm-LOBDP1) has been shown to increase kernel number andtotal kernel weight per plant (US Patent Application Publication Number2012/0079622).

(D) Enhancing yield-related traits in plants by modulating expression ina plant of a nucleic acid encoding a VIM1 (Variant in Methylation1)-like polypeptide or a VTC2-like (GDP-L-galactose phosphorylase)polypeptide or a DUF1685 polypeptide or an ARF6-like (Auxin ResponsiveFactor) polypeptide (WO 2012/038893).

(E) Modulating expression in a plant of a nucleic acid encoding aSte20-like polypeptide or a homologue thereof gives plants havingincreased yield relative to control plants (EP 2431472).

(F) Genes encoding nucleoside diphosphatase kinase (NDK) polypeptidesand homologs thereof for modifying the plant's root architecture (USPatent Application Publication Number 2009/0064373).

8. Genes that Confer Plant Digestibility.

(A) Altering the level of xylan present in the cell wall of a plant bymodulating expression of xylan synthase (U.S. Pat. No. 8,173,866).

In some embodiment the stacked trait may be a trait or event that hasreceived regulatory approval including but not limited to the events inTable 2A-1F.

TABLE 2A Triticum aestivum Wheat Event Company Description AP205CL BASFInc. Selection for a mutagenized version of the enzyme acetohydroxyacidsynthase (AHAS), also known as acetolactate synthase (ALS) oracetolactate pyruvate-lyase. AP602CL BASF Inc. Selection for amutagenized version of the enzyme acetohydroxyacid synthase (AHAS), alsoknown as acetolactate synthase (ALS) or acetolactate pyruvate-lyase.BW255-2, BW238-3 BASF Inc. Selection for a mutagenized version of theenzyme acetohydroxyacid synthase (AHAS), also known as acetolactatesynthase (ALS) or acetolactate pyruvate-lyase. BW7 BASF Inc. Toleranceto imidazolinone herbicides induced by chemical mutagenesis of theacetohydroxyacid synthase (AHAS) gene using sodium azide. MON71800Monsanto Company Glyphosate tolerant wheat variety produced by insertinga modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encodinggene from the soil bacterium Agrobacterium tumefaciens, strain CP4.SWP965001 Cyanamid Crop Selection for a mutagenized version of theProtection enzyme acetohydroxyacid synthase (AHAS), also known asacetolactate synthase (ALS) or acetolactate pyruvate-lyase. Teal 11ABASF Inc. Selection for a mutagenized version of the enzymeacetohydroxyacid synthase (AHAS), also known as acetolactate synthase(ALS) or acetolactate pyruvate-lyase. A2704-12, A2704-21, BayerCropScience Glufosinate ammonium herbicide tolerant A5547-35 (AventisCropScience soybean produced by inserting a modified (AgrEvo))phosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces viridochromogenes.

TABLE 2B Glycine max L. Soybean Event Company Description A5547-127Bayer CropScience Glufosinate ammonium herbicide tolerant (AventisCropScience soybean produced by inserting a modified (AgrEvo))phosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces viridochromogenes. BPS-CV127-9 BASF Inc. Theintroduced csr1-2 gene from Arabidopsis thaliana encodes anacetohydroxyacid synthase protein that confers tolerance toimidazolinone herbicides due to a point mutation that results in asingle amino acid substitution in which the serine residue at position653 is replaced by asparagine (S653N). DP-305423 Pioneer Hi-Bred Higholeic acid soybean produced by inserting International Inc. additionalcopies of a portion of the omega-6 desaturase encoding gene, gm-fad2-1resulting in silencing of the endogenous omega-6 desaturase gene(FAD2-1). DP356043 Pioneer Hi-Bred Soybean event with two herbicidetolerance International Inc. genes: glyphosate N-acetlytransferase,which detoxifies glyphosate, and a modified acetolactate synthase (ALS)gene which is tolerant to ALS-inhibiting herbicides. G94-1, G94-19, G168DuPont Canada High oleic acid soybean produced by inserting aAgricultural Products second copy of the fatty acid desaturase(GmFad2-1) encoding gene from soybean, which resulted in “silencing” ofthe endogenous host gene. GTS 40-3-2 Monsanto Company Glyphosatetolerant soybean variety produced by inserting a modified5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding gene fromthe soil bacterium Agrobacterium tumefaciens. GU262 Bayer CropScienceGlufosinate ammonium herbicide tolerant (Aventis soybean produced byinserting a modified CropScience(AgrEvo)) phosphinothricinacetyltransferase (PAT) encoding gene from the soil bacteriumStreptomyces viridochromogenes. MON87701 Monsanto Company Resistance tolepidopteran pests of soybean including velvetbean caterpillar(Anticarsia gemmatalis) and soybean looper (Pseudoplusia includens).MON87701 x Monsanto Company Glyphosate herbicide tolerance throughMON89788 expression of the EPSPS encoding gene from A. tumefaciensstrain CP4, and resistance to lepidopteran pests of soybean includingvelvetbean caterpillar (Anticarsia gemmatalis) and soybean looper(Pseudoplusia includens) via expression of the Cry1Ac encoding gene fromB. thuringiensis. MON89788 Monsanto Company Glyphosate-tolerant soybeanproduced by inserting a modified 5-enolpyruvylshikimate-3- phosphatesynthase (EPSPS) encoding aroA (epsps) gene from Agrobacteriumtumefaciens CP4. OT96-15 Agriculture & Agri-Food Low linolenic acidsoybean produced through Canada traditional cross-breeding toincorporate the novel trait from a naturally occurring fan1 gene mutantthat was selected for low linolenic acid. W62, W98 Bayer CropScienceGlufosinate ammonium herbicide tolerant (Aventis soybean produced byinserting a modified CropScience(AgrEvo)) phosphinothricinacetyltransferase (PAT) encoding gene from the soil bacteriumStreptomyces hygroscopicus.

TABLE 2C Helianthus annuus Sunflower Event Company Description X81359BASF Inc. Tolerance to imidazolinone herbicides by selection of anaturally occurring mutant.

TABLE 2D Medicago sativa Alfalfa Event Company Description J101,Monsanto Glyphosate herbicide tolerant alfalfa (lucerne) J163 Companyproduced by inserting a gene encoding the and Forage enzyme5-enolypyruvylshikimate-3-phosphate Genetics synthase (EPSPS) from theCP4 strain of International Agrobacterium tumefaciens.

TABLE 2E Oryza sativa Rice Event Company Description CL121, CL141, CFX51BASF Inc. Tolerance to the imidazolinone herbicide, imazethapyr, inducedby chemical mutagenesis of the acetolactate synthase (ALS) enzyme usingethyl methanesulfonate (EMS). IMINTA-1, IMINTA-4 BASF Inc. Tolerance toimidazolinone herbicides induced by chemical mutagenesis of theacetolactate synthase (ALS) enzyme using sodium azide. LLRICE06,LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerantrice produced by inserting a modified phosphinothricin acetyltransferase(PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus).LLRICE601 Bayer CropScience Glufosinate ammonium herbicide tolerant rice(Aventis produced by inserting a modified CropScience(AgrEvo))phosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces hygroscopicus). PWC16 BASF Inc. Tolerance to theimidazolinone herbicide, imazethapyr, induced by chemical mutagenesis ofthe acetolactate synthase (ALS) enzyme using ethyl methanesulfonate(EMS).

TABLE 2F Zea mays L. Maize Event Company Description 176 Syngenta Seeds,Inc. Insect-resistant maize produced by inserting the cry1Ab gene fromBacillus thuringiensis subsp. kurstaki. The genetic modification affordsresistance to attack by the European corn borer (ECB). 3751IR PioneerHi-Bred Selection of somaclonal variants by culture of InternationalInc. embryos on imidazolinone containing media. 676, 678, 680 PioneerHi-Bred Male-sterile and glufosinate ammonium herbicide InternationalInc. tolerant maize produced by inserting genes encoding DNA adeninemethylase and phosphinothricin acetyltransferase (PAT) from Escherichiacoli and Streptomyces viridochromogenes, respectively. B16 (DLL25)Dekalb Genetics Glufosinate ammonium herbicide tolerant maizeCorporation produced by inserting the gene encoding phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 (X4334CBR,Syngenta Seeds, Inc. Insect-resistant and herbicide tolerant maizeX4734CBR) produced by inserting the cry1Ab gene from Bacillusthuringiensis subsp. kurstaki, and the phosphinothricinN-acetyltransferase (PAT) encoding gene from S. viridochromogenes. BT11x GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicidetolerant maize produced by conventional cross breeding of parental linesBT11 (OECD unique identifier: SYN-BTO11-1) and GA21 (OECD uniqueidentifier: MON-OOO21-9). BT11 x MIR162 Syngenta Seeds, Inc. Stackedinsect resistant and herbicide tolerant maize produced by conventionalcross breeding of parental lines BT11 (OECD unique identifier:SYN-BTO11-1) and MIR162 (OECD unique identifier: SYN-IR162-4).Resistance to the European Corn Borer and tolerance to the herbicideglufosinate ammonium (Liberty) is derived from BT11, which contains thecry1Ab gene from Bacillus thuringiensis subsp. kurstaki, and thephosphinothricin N-acetyltransferase (PAT) encoding gene from S.viridochromogenes. Resistance to other lepidopteran pests, including H.zea, S. frugiperda, A. ipsilon, and S. albicosta, is derived fromMIR162, which contains the vip3Aa gene from Bacillus thuringiensisstrain AB88. BT11 x MIR162 x Syngenta Seeds, Inc. Bacillus thuringiensisCry1Ab delta-endotoxin MIR604 protein and the genetic material necessaryfor its production (via elements of vector pZO1502) in Event Bt11 corn(OECD Unique Identifier: SYN- BTO11-1) x Bacillus thuringiensis Vip3Aa20insecticidal protein and the genetic material necessary for itsproduction (via elements of vector pNOV1300) in Event MIR162 maize (OECDUnique Identifier: SYN-IR162-4) x modified Cry3A protein and the geneticmaterial necessary for its production (via elements of vector pZM26) inEvent MIR604 corn (OECD Unique Identifier: SYN-IR6O4-5). BT11 x SyngentaSeeds, Resistance to coleopteran pests, particularly corn rootworm pestsMIR162 x Inc. (Diabrotica spp.) and several lepidopteran pests of corn,including MIR604 x European corn borer (ECB, Ostrinia nubilalis), cornearworm GA21 (CEW, Helicoverpa zea), fall army worm (FAW, Spodopterafrugiperda), and black cutworm (BCW, Agrotis ipsilon); tolerance toglyphosate and glufosinate-ammonium containing herbicides. BT11 xSyngenta Seeds, Stacked insect resistant and herbicide tolerant maizeproduced MIR604 Inc. by conventional cross breeding of parental linesBT11 (OECD unique identifier: SYN-BTO11-1) and MIR604 (OECD uniqueidentifier: SYN-IR6O5-5). Resistance to the European Corn Borer andtolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N- acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm-resistance isderived from MIR604 which contains the mcry3A gene from Bacillusthuringiensis. BT11 x Syngenta Seeds, Stacked insect resistant andherbicide tolerant maize produced MIR604 x Inc. by conventional crossbreeding of parental lines BT11 (OECD GA21 unique identifier:SYN-BTO11-1), MIR604 (OECD unique identifier: SYN-IR6O5-5) and GA21(OECD unique identifier: MON-OOO21-9). Resistance to the European CornBorer and tolerance to the herbicide glufosinate ammonium (Liberty) isderived from BT11, which contains the cry1Ab gene from Bacillusthuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. Cornrootworm-resistance is derived from MIR604 which contains the mcry3Agene from Bacillus thuringiensis. Tolerance to glyphosate herbicide isderived from GA21 which contains a modified EPSPS gene from maize.CBH-351 Aventis Insect-resistant and glufosinate ammonium herbicidetolerant CropScience maize developed by inserting genes encoding Cry9Cprotein from Bacillus thuringiensis subsp tolworthi and phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. DAS- DOWAgroSciences Lepidopteran insect resistant and glufosinate ammonium06275-8 LLC herbicide-tolerant maize variety produced by inserting thecry1F gene from Bacillus thuringiensis var aizawai and thephosphinothricin acetyltransferase (PAT) from Streptomyceshygroscopicus. DAS- DOW AgroSciences Corn rootworm-resistant maizeproduced by inserting the 59122-7 LLC and Pioneer Hi- cry34Ab1 andcry35Ab1 genes from Bacillus thuringiensis strain Bred InternationalPS149B1. The PAT encoding gene from Streptomyces Inc. viridochromogeneswas introduced as a selectable marker. DAS- DOW AgroSciences Stackedinsect resistant and herbicide tolerant maize produced 59122-7 x LLC andPioneer Hi- by conventional cross breeding of parental lines DAS-59122-7NK603 Bred International (OECD unique identifier: DAS-59122-7) withNK603 (OECD Inc. unique identifier: MON-OO6O3-6). Cornrootworm-resistance is derived from DAS-59122-7 which contains thecry34Ab1 and cry35Ab1 genes from Bacillus thuringiensis strain PS149B1.Tolerance to glyphosate herbicide is derived from NK603. DAS-59122-7 DOWAgroSciences Stacked insect resistant and herbicide tolerant maizeproduced x TC1507 x LLC and Pioneer by conventional cross breeding ofparental lines DAS-59122-7 NK603 Hi-Bred (OECD unique identifier:DAS-59122-7) and TC1507 (OECD International Inc. unique identifier:DAS-O15O7-1) with NK603 (OECD unique identifier: MON-OO6O3-6). Cornrootworm-resistance is derived from DAS-59122-7 which contains thecry34Ab1 and cry35Ab1 genes from Bacillus thuringiensis strain PS149B1.Lepidopteran resistance and tolerance to glufosinate ammonium herbicideis derived from TC1507. Tolerance to glyphosate herbicide is derivedfrom NK603. DBT418 Dekalb Genetics Insect-resistant and glufosinateammonium herbicide tolerant Corporation maize developed by insertinggenes encoding Cry1AC protein from Bacillus thuringiensis subsp kurstakiand phosphinothricin acetyltransferase (PAT) from Streptomyceshygroscopicus DK404SR BASF Inc. Somaclonal variants with a modifiedacetyl-CoA-carboxylase (ACCase) were selected by culture of embryos onsethoxydim enriched medium. Event 3272 Syngenta Seeds, Maize lineexpressing a heat stable alpha-amylase gene Inc. amy797E for use in thedry-grind ethanol process. The phosphomannose isomerase gene from E.coli was used as a selectable marker. Event 98140 Pioneer Hi-Bred Maizeevent expressing tolerance to glyphosate herbicide, via InternationalInc. expression of a modified bacterial glyphosate N- acetlytransferase,and ALS-inhibiting herbicides, vial expression of a modified form of themaize acetolactate synthase enzyme. EXP1910IT Syngenta Seeds, Toleranceto the imidazolinone herbicide, imazethapyr, Inc. (formerly induced bychemical mutagenesis of the acetolactate synthase Zeneca Seeds) (ALS)enzyme using ethyl methanesulfonate (EMS). GA21 Syngenta Seeds,Introduction, by particle bombardment, of a modified 5- Inc. (formerlyenolpyruvyl shikimate-3-phosphate synthase (EPSPS), an Zeneca Seeds)enzyme involved in the shikimate biochemical pathway for the productionof the aromatic amino acids. GA21 x Monsanto Company Stacked insectresistant and herbicide tolerant corn hybrid MON810 derived fromconventional cross-breeding of the parental lines GA21 (OECD identifier:MON-OOO21-9) and MON810 (OECD identifier: MON-OO81O-6). IT PioneerHi-Bred Tolerance to the imidazolinone herbicide, imazethapyr, wasInternational Inc. obtained by in vitro selection of somaclonalvariants. LY038 Monsanto Company Altered amino acid composition,specifically elevated levels of lysine, through the introduction of thecordapA gene, derived from Corynebacterium glutamicum, encoding theenzyme dihydrodipicolinate synthase (cDHDPS). MIR162 Syngenta Seeds,Insect-resistant maize event expressing a Vip3A protein from Inc.Bacillus thuringiensis and the Escherichia coli PMI selectable markerMIR604 Syngenta Seeds, Corn rootworm resistant maize produced bytransformation Inc. with a modified cry3A gene. The phosphomannoseisomerase gene from E. coli was used as a selectable marker. MIR604 xSyngenta Stacked insect resistant and herbicide tolerant maize producedby GA21 Seeds, Inc. conventional cross breeding of parental lines MIR604(OECD unique identifier: SYN-IR6O5-5) and GA21 (OECD unique identifier:MON- OOO21-9). Corn rootworm-resistance is derived from MIR604 whichcontains the mcry3A gene from Bacillus thuringiensis. Tolerance toglyphosate herbicide is derived from GA21. MON80100 MonsantoInsect-resistant maize produced by inserting the cry1Ab gene fromCompany Bacillus thuringiensis subsp. kurstaki. The genetic modificationaffords resistance to attack by the European corn borer (ECB). MON802Monsanto Insect-resistant and glyphosate herbicide tolerant maizeproduced Company by inserting the genes encoding the Cry1Ab protein fromBacillus thuringiensis and the 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) from A. tumefaciens strain CP4. MON809 Pioneer Hi-Resistance to European corn borer (Ostrinia nubilalis) by Bredintroduction of a synthetic cry1Ab gene. Glyphosate resistance viaInternational introduction of the bacterial version of a plant enzyme,5-enolpyruvyl Inc. shikimate-3-phosphate synthase (EPSPS). MON810Monsanto Insect-resistant maize produced by inserting a truncated formof the Company cry1Ab gene from Bacillus thuringiensis subsp. kurstakiHD-1. The genetic modification affords resistance to attack by theEuropean corn borer (ECB). MON810 x Monsanto Stacked insect resistantand enhanced lysine content maize derived LY038 Company fromconventional cross-breeding of the parental lines MON810 (OECDidentifier: MON-OO81O-6) and LY038 (OECD identifier: REN-OOO38-3).MON810 x Monsanto Stacked insect resistant and glyphosate tolerant maizederived from MON88017 Company conventional cross-breeding of theparental lines MON810 (OECD identifier: MON-OO81O-6) and MON88017 (OECDidentifier:MON- 88O17-3). European corn borer (ECB) resistance isderived from a truncated form of the cry1Ab gene from Bacillusthuringiensis subsp. kurstaki HD-1 present in MON810. Corn rootwormresistance is derived from the cry3Bb1 gene from Bacillus thuringiensissubspecies kumamotoensis strain EG4691 present in MON88017. Glyphosatetolerance is derived from a 5-enolpyruvylshikimate-3- phosphate synthase(EPSPS) encoding gene from Agrobacterium tumefaciens strain CP4 presentin MON88017. MON832 Monsanto Introduction, by particle bombardment, ofglyphosate oxidase (GOX) Company and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in theshikimate biochemical pathway for the production of the aromatic aminoacids. MON863 Monsanto Corn root worm resistant maize produced byinserting the cry3Bb1 Company gene from Bacillus thuringiensis subsp.kumamotoensis. MON863 x Monsanto Stacked insect resistant corn hybridderived from conventional MON810 Company cross-breeding of the parentallines MON863 (OECD identifier: MON-OO863-5) and MON810 (OECD identifier:MON-OO810-6) MON863 x Monsanto Stacked insect resistant and herbicidetolerant corn hybrid derived MON810 x Company from conventionalcross-breeding of the stacked hybrid MON- NK603 OO863-5 x MON-OO81O-6and NK603 (OECD identifier: MON- OO6O3-6). MON863 x Monsanto CompanyStacked insect resistant and herbicide tolerant NK603 corn hybridderived from conventional cross- breeding of the parental lines MON863(OECD identifier: MON-OO863-5) and NK603 (OECD identifier: MON-OO6O3-6).MON87460 Monsanto Company MON 87460 was developed to provide reducedyield loss under water-limited conditions compared to conventionalmaize. Efficacy in MON 87460 is derived by expression of the insertedBacillus subtilis cold shock protein B (CspB). MON88017 Monsanto CompanyCorn rootworm-resistant maize produced by inserting the cry3Bb1 genefrom Bacillus thuringiensis subspecies kumamotoensis strain EG4691.Glyphosate tolerance derived by inserting a5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene fromAgrobacterium tumefaciens strain CP4. MON89034 Monsanto Company Maizeevent expressing two different insecticidal proteins from Bacillusthuringiensis providing resistance to number of lepidopteran pests.MON89034 Monsanto Company Stacked insect resistant and glyphosatetolerant x maize derived from conventional cross-breeding MON88017 ofthe parental lines MON89034 (OECD identifier: MON-89O34-3) and MON88017(OECD identifier: MON-88O17-3). Resistance to Lepidopteran insects isderived from two cry genes present in MON89043. Corn rootworm resistanceis derived from a single cry genes and glyphosate tolerance is derivedfrom the 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encodinggene from Agrobacterium tumefaciens present in MON88017. MON89034Monsanto Company Stacked insect resistant and herbicide tolerant x NK603maize produced by conventional cross breeding of parental lines MON89034(OECD identifier: MON-89O34-3) with NK603 (OECD unique identifier:MON-OO6O3-6). Resistance to Lepidopteran insects is derived from two crygenes present in MON89043. Tolerance to glyphosate herbicide is derivedfrom NK603. MON89034 Monsanto Company and Mycogen Stacked insectresistant and herbicide tolerant x TC1507 x Seeds c/o Dow AgroSciencesLLC maize produced by conventional cross breeding MON88017 of parentallines: MON89034, TC1507, x DAS- MON88017, and DAS-59122. Resistance tothe 59122-7 above-ground and below-ground insect pests and tolerance toglyphosate and glufosinate- ammonium containing herbicides. MS3 BayerCropScience (Aventis Male sterility caused by expression of the barnaseCropScience(AgrEvo)) ribonuclease gene from Bacillus amyloliquefaciens;PPT resistance was via PPT- acetyltransferase (PAT). MS6 BayerCropScience (Aventis Male sterility caused by expression of the barnaseCropScience(AgrEvo)) ribonuclease gene from Bacillus amyloliquefaciens;PPT resistance was via PPT- acetyltransferase (PAT). NK603 MonsantoCompany Introduction, by particle bombardment, of a modified5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), an enzyme involvedin the shikimate biochemical pathway for the production of the aromaticamino acids. NK603 x Monsanto Company Stacked insect resistant andherbicide tolerant MON810 corn hybrid derived from conventional cross-breeding of the parental lines NK603 (OECD identifier: MON-OO6O3-6) andMON810 (OECD identifier: MON-OO81O-6). NK603 x Monsanto Company Stackedglufosinate ammonium and glyphosate T25 herbicide tolerant maize hybridderived from conventional cross-breeding of the parental lines NK603(OECD identifier: MON-OO6O3-6) and T25 (OECD identifier: ACS-ZM003-2).T14, T25 Bayer CropScience (Aventis Glufosinate herbicide tolerant maizeproduced by CropScience(AgrEvo)) inserting the phosphinothricinN-acetyltransferase (PAT) encoding gene from the aerobic actinomyceteStreptomyces viridochromogenes. T25 x Bayer CropScience (Aventis Stackedinsect resistant and herbicide tolerant MON810 CropScience(AgrEvo)) cornhybrid derived from conventional cross- breeding of the parental linesT25 (OECD identifier: ACS-ZMOO3-2) and MON810 (OECD identifier:MON-OO81O-6). TC1507 Mycogen (c/o Dow AgroSciences); Insect-resistantand glufosinate ammonium Pioneer (c/o DuPont) herbicide tolerant maizeproduced by inserting the cry1F gene from Bacillus thuringiensis var.aizawai and the phosphinothricin N- acetyltransferase encoding gene fromStreptomyces viridochromogenes. TC1507 x DOW AgroSciences LLC andStacked insect resistant and herbicide tolerant DAS- Pioneer Hi-BredInternational Inc. maize produced by conventional cross breeding of59122-7 parental lines TC1507 (OECD unique identifier: DAS-O15O7-1) withDAS-59122-7 (OECD unique identifier: DAS-59122-7). Resistance tolepidopteran insects is derived from TC1507 due the presence of thecry1F gene from Bacillus thuringiensis var. aizawai. Corn rootworm-resistance is derived from DAS-59122-7 which contains the cry34Ab1 andcry35Ab1 genes from Bacillus thuringiensis strain PS149B1. Tolerance toglufosinate ammonium herbicide is derived from TC1507 from thephosphinothricin N- acetyltransferase encoding gene from Streptomycesviridochromogenes. TC1507 x DOW AgroSciences LLC Stacked insectresistant and herbicide tolerant NK603 corn hybrid derived fromconventional cross- breeding of the parental lines 1507 (OECDidentifier: DAS-O15O7-1) and NK603 (OECD identifier: MON-OO6O3-6).

Other events with regulatory approval are well known to one skilled inthe art and can be found at the Center for Environmental Risk Assessment(cera-gmc.org/?action=gm_crop_database, which can be accessed using thewww prefix).

Gene Silencing

In some embodiments the stacked trait may be in the form of silencing ofone or more polynucleotides of interest resulting in suppression of oneor more target pest polypeptides. In some embodiments the silencing isachieved through the use of a suppression DNA construct.

In some embodiments one or more of the PHI-4 polypeptides or fragmentsor variants thereof may be stacked with one or more polynucleotidesencoding one or more polypeptides having insecticidal activity oragronomic traits as set forth supra and optionally may further includeone or more polynucleotides providing for gene silencing of one or moretarget polynucleotides as discussed infra.

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The term“suppression” includes lower, reduce, decline, decrease, inhibit,eliminate and prevent. “Silencing” or “gene silencing” does not specifymechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50% or anyinteger between 51% and 100% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.

“Antisense RNA” refers to an RNA transcript that is complementary to allor part of a target primary transcript or mRNA and that blocks theexpression of a target isolated nucleic acid fragment (U.S. Pat. No.5,107,065). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns or the coding sequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target protein. “Sense” RNArefers to RNA transcript that includes the mRNA and can be translatedinto protein within a cell or in vitro. Cosuppression constructs inplants have been previously designed by focusing on overexpression of anucleic acid sequence having homology to a native mRNA, in the senseorientation, which results in the reduction of all RNA having homologyto the overexpressed sequence (see, Vaucheret, et al. (1998) Plant J.16:651-659 and Gura, (2000) Nature 404:804-808).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication WO1998/36083).

Recent work has described the use of “hairpin” structures thatincorporate all, or part, of an mRNA encoding sequence in acomplementary orientation that results in a potential “stem-loop”structure for the expressed RNA (PCT Publication Number WO 1999/53050).In this case the stem is formed by polynucleotides corresponding to thegene of interest inserted in either sense or anti-sense orientation withrespect to the promoter and the loop is formed by some polynucleotidesof the gene of interest, which do not have a complement in theconstruct. This increases the frequency of cosuppression or silencing inthe recovered transgenic plants. For review of hairpin suppression see,Wesley, et al., (2003) Methods in Molecular Biology, Plant FunctionalGenomics: Methods and Protocols 236:273-286.

A construct where the stem is formed by at least 30 nucleotides from agene to be suppressed and the loop is formed by a random nucleotidesequence has also effectively been used for suppression (WO 1999/61632).

The use of poly-T and poly-A sequences to generate the stem in thestem-loop structure has also been described (WO 2002/00894).

Yet another variation includes using synthetic repeats to promoteformation of a stem in the stem-loop structure. Transgenic organismsprepared with such recombinant DNA fragments have been shown to havereduced levels of the protein encoded by the nucleotide fragment formingthe loop as described in PCT Publication Number WO 2002/00904.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire, et al., (1999) TrendsGenet. 15:358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA of viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein, et al., (2001) Nature 409:363).Short interfering RNAs derived from dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes (Elbashir, et aL, (2001) Genes Dev. 15:188). Dicer has alsobeen implicated in the excision of 21- and 22-nucleotide small temporalRNAs (stRNAs) from precursor RNA of conserved structure that areimplicated in translational control (Hutvagner, et al., (2001) Science293:834). The RNAi response also features an endonuclease complex,commonly referred to as an RNA-induced silencing complex (RISC), whichmediates cleavage of single-stranded RNA having sequence complementarityto the antisense strand of the siRNA duplex. Cleavage of the target RNAtakes place in the middle of the region complementary to the antisensestrand of the siRNA duplex (Elbashir, et al., (2001) Genes Dev. 15:188).In addition, RNA interference can also involve small RNA (e.g., miRNA)mediated gene silencing, presumably through cellular mechanisms thatregulate chromatin structure and thereby prevent transcription of targetgene sequences (see, e.g., Allshire, (2002) Science 297:1818-1819;Volpe, et al., (2002) Science 297:1833-1837; Jenuwein, (2002) Science297:2215-2218; and Hall, et al., (2002) Science 297:2232-2237). As such,miRNA molecules of the invention can be used to mediate gene silencingvia interaction with RNA transcripts or alternately by interaction withparticular gene sequences, wherein such interaction results in genesilencing either at the transcriptional or post-transcriptional level.

Methods and compositions are further provided which allow for anincrease in RNAi produced from the silencing element. In suchembodiments, the methods and compositions employ a first polynucleotidecomprising a silencing element for a target pest sequence operablylinked to a promoter active in the plant cell; and, a secondpolynucleotide comprising a suppressor enhancer element comprising thetarget pest sequence or an active variant or fragment thereof operablylinked to a promoter active in the plant cell. The combined expressionof the silencing element with suppressor enhancer element leads to anincreased amplification of the inhibitory RNA produced from thesilencing element over that achievable with only the expression of thesilencing element alone. In addition to the increased amplification ofthe specific RNAi species itself, the methods and compositions furtherallow for the production of a diverse population of RNAi species thatcan enhance the effectiveness of disrupting target gene expression. Assuch, when the suppressor enhancer element is expressed in a plant cellin combination with the silencing element, the methods and compositioncan allow for the systemic production of RNAi throughout the plant; theproduction of greater amounts of RNAi than would be observed with justthe silencing element construct alone; and, the improved loading of RNAiinto the phloem of the plant, thus providing better control of phloemfeeding insects by an RNAi approach. Thus, the various methods andcompositions provide improved methods for the delivery of inhibitory RNAto the target organism. See, for example, US 2009/0188008.

As used herein, a “suppressor enhancer element” comprises apolynucleotide comprising the target sequence to be suppressed or anactive fragment or variant thereof. It is recognize that the suppressorenhancer element need not be identical to the target sequence, butrather, the suppressor enhancer element can comprise a variant of thetarget sequence, so long as the suppressor enhancer element hassufficient sequence identity to the target sequence to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element. Similarly,the suppressor enhancer element can comprise a fragment of the targetsequence, wherein the fragment is of sufficient length to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element.

It is recognized that multiple suppressor enhancer elements from thesame target sequence or from different target sequences, or fromdifferent regions of the same target sequence can be employed. Forexample, the suppressor enhancer elements employed can comprisefragments of the target sequence derived from different region of thetarget sequence (i.e., from the 3′UTR, coding sequence, intron, and/or5′UTR). Further, the suppressor enhancer element can be contained in anexpression cassette, as described elsewhere herein, and in specificembodiments, the suppressor enhancer element is on the same or on adifferent DNA vector or construct as the silencing element. Thesuppressor enhancer element can be operably linked to a promoter asdisclosed herein. It is recognized that the suppressor enhancer elementcan be expressed constitutively or alternatively, it may be produced ina stage-specific manner employing the various inducible ortissue-preferred or developmentally regulated promoters that arediscussed elsewhere herein.

In specific embodiments, employing both a silencing element and thesuppressor enhancer element the systemic production of RNAi occursthroughout the entire plant. In further embodiments, the plant or plantparts of the invention have an improved loading of RNAi into the phloemof the plant than would be observed with the expression of the silencingelement construct alone and, thus provide better control of phloemfeeding insects by an RNAi approach. In specific embodiments, theplants, plant parts, and plant cells of the invention can further becharacterized as allowing for the production of a diversity of RNAispecies that can enhance the effectiveness of disrupting target geneexpression.

In specific embodiments, the combined expression of the silencingelement and the suppressor enhancer element increases the concentrationof the inhibitory RNA in the plant cell, plant, plant part, plant tissueor phloem over the level that is achieved when the silencing element isexpressed alone.

As used herein, an “increased level of inhibitory RNA” comprises anystatistically significant increase in the level of RNAi produced in aplant having the combined expression when compared to an appropriatecontrol plant. For example, an increase in the level of RNAi in theplant, plant part or the plant cell can comprise at least about a 1%,about a 1%-5%, about a 5%-10%, about a 10%-20%, about a 20%-30%, about a30%-40%, about a 40%-50%, about a 50%-60%, about 60-70%, about 70%-80%,about a 80%-90%, about a 90%-100% or greater increase in the level ofRNAi in the plant, plant part, plant cell, or phloem when compared to anappropriate control. In other embodiments, the increase in the level ofRNAi in the plant, plant part, plant cell, or phloem can comprise atleast about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold,about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 fold-70fold, about 70 fold-80 fold, about a 80 fold-90 fold, about a 90fold-100 fold or greater increase in the level of RNAi in the plant,plant part, plant cell or phloem when compared to an appropriatecontrol. Examples of combined expression of the silencing element withsuppressor enhancer element for the control of Stinkbugs and Lygus canbe found in US 2011/0301223 and US 2009/0192117.

Some embodiments relate to down-regulation of expression of target genesin insect pest species by interfering ribonucleic acid (RNA) molecules.WO 2007/074405 describes methods of inhibiting expression of targetgenes in invertebrate pests including Colorado potato beetle. WO2005/110068 describes methods of inhibiting expression of target genesin invertebrate pests including in particular Western corn rootworm as ameans to control insect infestation. Furthermore, WO 2009/091864describes compositions and methods for the suppression of target genesfrom insect pest species including pests from the Lygus genus. Nucleicacid molecules including RNAi for targeting the vacuolar ATPase Hsubunit, useful for controlling a coleopteran pest population andinfestation as described in US Patent Application Publication2012/0198586. WO 2012/055982 describes ribonucleic acid (RNA or doublestranded RNA) that inhibits or down regulates the expression of a targetgene that encodes: an insect ribosomal protein such as the ribosomalprotein L19, the ribosomal protein L40 or the ribosomal protein S27A; aninsect proteasome subunit such as the Rpn6 protein, the Pros 25, theRpn2 protein, the proteasome beta 1 subunit protein or the Pros beta 2protein; an insect β-coatomer of the COPI vesicle, the γ-coatomer of theCOPI vesicle, the 3′-coatomer protein or the ζ-coatomer of the COPIvesicle; an insect Tetraspanine 2 A protein which is a putativetransmembrane domain protein; an insect protein belonging to the actinfamily such as Actin 5C; an insect ubiquitin-5E protein; an insect Sec23protein which is a GTPase activator involved in intracellular proteintransport; an insect crinkled protein which is an unconventional myosinwhich is involved in motor activity; an insect crooked neck proteinwhich is involved in the regulation of nuclear alternative mRNAsplicing; an insect vacuolar H₊-ATPase G-subunit protein; and an insectTbp-1 such as Tat-binding protein. US Patent Application Publications2012/029750, US 20120297501, and 2012/0322660 describe interferingribonucleic acids (RNA or double stranded RNA) that functions uponuptake by an insect pest species to down-regulate expression of a targetgene in said insect pest, wherein the RNA comprises at least onesilencing element wherein the silencing element is a region ofdouble-stranded RNA comprising annealed complementary strands, onestrand of which comprises or consists of a sequence of nucleotides whichis at least partially complementary to a target nucleotide sequencewithin the target gene. US Patent Application Publication 2012/0164205describe potential targets for interfering double stranded ribonucleicacids for inhibiting invertebrate pests including: a Chd3 HomologousSequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPaseHomologous Sequence, a EF1α Homologous Sequence, a 26S ProteosomeSubunit p28 Homologous Sequence, a Juvenile Hormone Epoxide HydrolaseHomologous Sequence, a Swelling Dependent Chloride Channel ProteinHomologous Sequence, a Glucose-6-Phosphate 1-Dehydrogenase ProteinHomologous Sequence, an Act42A Protein Homologous Sequence, aADP-Ribosylation Factor 1 Homologous Sequence, a Transcription FactorIIB Protein Homologous Sequence, a Chitinase Homologous Sequences, aUbiquitin Conjugating Enzyme Homologous Sequence, aGlyceraldehyde-3-Phosphate Dehydrogenase Homologous Sequence, anUbiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog,and an Alpha Tubuliln Homologous Sequence.

Use in Pesticidal Control

General methods for employing strains comprising a nucleic acid sequenceof the embodiments, or a variant thereof, in pesticide control or inengineering other organisms as pesticidal agents are known in the art.See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the PHI-4 polypeptide,and desirably, provide for improved protection of the pesticide fromenvironmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Pseudomonas chlororaphis, Serratia marcescens,Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,Clavibacter xyli and Azotobacter vinelandii and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms. Host organismsof particular interest include yeast, such as Rhodotorula spp.,Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae),Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp.(such as P. aeruginosa, P. fluorescens, P. chlororaphis), Erwinia spp.,and Flavobacterium spp., and other such organisms, includingAgrobacterium tumefaciens, E. coli, Bacillus subtilis, Bacillus cereusand the like.

Genes encoding the PHI-4 polypeptides of the embodiments can beintroduced into microorganisms that multiply on plants (epiphytes) todeliver PHI-4 polypeptides to potential target pests. Epiphytes, forexample, can be gram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain that colonizes roots can be isolated from roots of a plant (see,for example, Handelsman, et al., (1991) Appl. Environ. Microbiol.56:713-718). Genes encoding the PHI-4 polypeptides of the embodimentscan be introduced into a root-colonizing Bacillus cereus by standardmethods known in the art.

Genes encoding PHI-4 polypeptides can be introduced, for example, intothe root-colonizing Bacillus by means of electro transformation.Specifically, genes encoding the PHI-4 polypeptides can be cloned into ashuttle vector, for example, pHT3101 (Lerecius, et al., (1989) FEMSMicrobiol. Letts. 60:211-218. The shuttle vector pHT3101 containing thecoding sequence for the particular PHI-4 polypeptide gene can, forexample, be transformed into the root-colonizing Bacillus by means ofelectroporation (Lerecius, et al., (1989) FEMS Microbiol. Letts.60:211-218).

Expression systems can be designed so that PHI-4 polypeptides aresecreted outside the cytoplasm of gram-negative bacteria, such as E.coli, for example. Advantages of having PHI-4 polypeptides secreted are:(1) avoidance of potential cytotoxic effects of the PHI-4 polypeptideexpressed; and (2) improvement in the efficiency of purification of thePHI-4 polypeptide, including, but not limited to, increased efficiencyin the recovery and purification of the protein per volume cell brothand decreased time and/or costs of recovery and purification per unitprotein.

PHI-4 polypeptides can be made to be secreted in E. coli, for example,by fusing an appropriate E. coli signal peptide to the amino-terminalend of the PHI-4 polypeptide. Signal peptides recognized by E. coli canbe found in proteins already known to be secreted in E. coli, forexample the OmpA protein (Ghrayeb, et al., (1984) EMBO J, 3:2437-2442).OmpA is a major protein of the E. coli outer membrane, and thus itssignal peptide is thought to be efficient in the translocation process.Also, the OmpA signal peptide does not need to be modified beforeprocessing as may be the case for other signal peptides, for examplelipoprotein signal peptide (Duffaud, et al., (1987) Meth. Enzymol.153:492).

PHI-4 polypeptides of the embodiments can be fermented in a bacterialhost and the resulting bacteria processed and used as a microbial sprayin the same manner that Bt strains have been used as insecticidalsprays. In the case of a PHI-4 polypeptide that is secreted fromBacillus, the secretion signal is removed or mutated using proceduresknown in the art. Such mutations and/or deletions prevent secretion ofthe PHI-4 polypeptide into the growth medium during the fermentationprocess. The PHI-4 polypeptides are retained within the cell, and thecells are then processed to yield the encapsulated PHI-4 polypeptides.Any suitable microorganism can be used for this purpose. Pseudomonas hasbeen used to express Bt toxins as encapsulated proteins and theresulting cells processed and sprayed as an insecticide (Gaertner, etal., (1993), in: Advanced Engineered Pesticides, ed. Kim).

Alternatively, the PHI-4 polypeptides are produced by introducing aheterologous gene into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated PHI-4 polypeptides may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein.

Pesticidal Compositions

In some embodiments the active ingredients can be applied in the form ofcompositions and can be applied to the crop area or plant to be treated,simultaneously or in succession, with other compounds. These compoundscan be fertilizers, weed killers, cryoprotectants, surfactants,detergents, pesticidal soaps, dormant oils, polymers, and/ortime-release or biodegradable carrier formulations that permit long-termdosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient or an agrochemical compositionthat contains at least one of the PHI-4 polypeptides produced by thebacterial strains include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution, or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, dipteran, heteropteran, nematode, hemiptera, orcoleopteran pests may be killed or reduced in numbers in a given area bythe methods of the disclosure, or may be prophylactically applied to anenvironmental area to prevent infestation by a susceptible pest.Preferably the pest ingests, or is contacted with, apesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations may also vary with respect to climatic conditions,environmental considerations, and/or frequency of application and/orseverity of pest infestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference. The plants can also be treated with one or more chemicalcompositions, including one or more herbicide, insecticides, orfungicides. Exemplary chemical compositions include: Fruits/VegetablesHerbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin,Simazine, Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan,Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron,Indaziflam; Fruits/Vegetables Insecticides: Aldicarb, Bacillusthuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin,Deltamethrin, Diazinon, Malathion, Abamectin,Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin,Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide,Thiacloprid, Dinotefuran, Fluacrypyrim, Tolfenpyrad, Clothianidin,Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr,Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid,Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen,Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram,Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb,Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid,Hexthiazox, Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon,Bromoxynil, loxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop,Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron,Triasulfuron, Flucarbazone, lodosulfuron, Propoxycarbazone, Picolinafen,Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, ThifensulfuronMethyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole,Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides:Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil,Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor,Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione,Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone,Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos,Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin,Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide,Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin,Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin,Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid,Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; MaizeFungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole,Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron,Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron,Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac,Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron,Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac,Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione,Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon,Fenitrothion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin,Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid,Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole,Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin,Chlorpyriphos, Cartap, Methamidophos, Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:Carbofuran, Organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,3-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

In some embodiments the herbicide is Atrazine, Bromacil, Diuron,Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron,Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron,Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin,Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

In some embodiments the insecticide is Esfenvalerate,Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl, or combinationsthereof.

Pesticidal and Insecticidal Activity

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera, and Hemiptera.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery, ornamentals, food andfiber, public and animal health, domestic and commercial structure,household and stored product pests.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers, and heliothines in the family NoctuidaeSpodoptera frugiperda JE Smith (fall armyworm); S. exigua Hubner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hubner (cotton leaf worm); Trichoplusia niHubner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hubner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hubner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hubner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guen6e (rice leafroller); Desmia funeralis Hubner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hubner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hubner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms, and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRosslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermller (European grape vine moth);Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHubner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hubner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail, orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée(omnivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenée; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae, and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles,and leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smith &Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (corn flea beetle); Colaspisbrunnea Fabricius (grape colaspis); Oulema melanopus Linnaeus (cerealleaf beetle); Zygogramma exclamationis Fabricius (sunflower beetle));beetles from the family Coccinellidae (including, but not limited to:Epilachna varivestis Mulsant (Mexican bean beetle)); chafers and otherbeetles from the family Scarabaeidae (including, but not limited to:Popillia japonica Newman (Japanese beetle); Cyclocephala borealis Arrow(northern masked chafer, white grub); C. immaculata Olivier (southernmasked chafer, white grub); Rhizotrogus majalis Razoumowsky (Europeanchafer); Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosusDe Geer (carrot beetle)); carpet beetles from the family Dermestidae;wireworms from the family Elateridae, Eleodes spp., Melanotus spp.;Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolusspp.; bark beetles from the family Scolytidae and beetles from thefamily Tenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Géhin (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly);and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp.; and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds); and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae, Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae, and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid); and T. citricida Kirkaldy (browncitrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande(pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly,sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleafwhitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodesabutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood(greenhouse whitefly); Empoasca fabae Harris (potato leafhopper);Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestesquadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler(green leafhopper); N. nigropictus Stal (rice leafhopper); Nilaparvatalugens Stal (brown planthopper); Peregrinus maidis Ashmead (cornplanthopper); Sogatella furcifera Horvath (white-backed planthopper);Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee(white apple leafhopper); Erythroneoura spp. (grape leafhoppers);Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasiMaskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock(San Jose scale); Planococcus citri Risso (citrus mealybug);Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster(pear psylla); Trioza diospyri Ashmead (persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schiffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.;and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Müller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e. dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick); and scab and itch mites in thefamilies Psoroptidae, Pyemotidae, and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Additional arthropod pests covered include: spiders in the order Araneaesuch as Loxosceles reclusa Gertsch & Mulaik (brown recluse spider); andthe Latrodectus mactans Fabricius (black widow spider); and centipedesin the order Scutigeromorpha such as Scutigera coleoptrata Linnaeus(house centipede).

Insect pest of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria—Bean plataspid), and the family Cydnidae(Scaptocoris castanea—Root stink bug); and Lepidoptera species includingbut not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker; and velvet beancaterpillar e.g., Anticarsia gemmatalis Hubner.

Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang, (1990) J. Econ. Entomol.83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone,et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety. Generally, the protein is mixed and used in feeding assays.See, for example Marrone, et al., (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Seed Treatment

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases. Seed material canbe treated, typically surface treated, with a composition comprisingcombinations of chemical or biological herbicides, herbicide safeners,insecticides, fungicides, germination inhibitors and enhancers,nutrients, plant growth regulators and activators, bactericides,nematocides, avicides and/or molluscicides. These compounds aretypically formulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. The coatings may be applied by impregnating propagationmaterial with a liquid formulation or by coating with a combined wet ordry formulation. Examples of the various types of compounds that may beused as seed treatments are provided in The Pesticide Manual: A WorldCompendium, C. D. S. Tomlin Ed., Published by the British CropProduction Council, which is hereby incorporated by reference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species),bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA registrationnumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

Methods for Inhibiting Growth or Killing an Insect Pest and Controllingan Insect Population

In some embodiments methods are provided for inhibiting growth orkilling an insect pest, comprising contacting the insect pest with aninsecticidally-effective amount of a recombinant PHI-4 polypeptide. Insome embodiments methods are provided for inhibiting growth or killingan insect pest, comprising contacting the insect pest with aninsecticidally-effective amount of a recombinant pesticidal protein ofSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NOs: 51-819 or avariant thereof.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant PHI-4 polypeptide. Insome embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant pesticidal protein ofSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NOs: 51-819 or avariant thereof. As used herein, by “controlling a pest population” or“controls a pest” is intended any effect on a pest that results inlimiting the damage that the pest causes. Controlling a pest includes,but is not limited to, killing the pest, inhibiting development of thepest, altering fertility or growth of the pest in such a manner that thepest provides less damage to the plant, decreasing the number ofoffspring produced, producing less fit pests, producing pests moresusceptible to predator attack or deterring the pests from eating theplant.

In some embodiments methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population with an insecticidally-effective amount of arecombinant PHI-4 polypeptide. In some embodiments methods are providedfor controlling an insect pest population resistant to a pesticidalprotein, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant pesticidal protein ofSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NOs: 51-819 or avariant thereof.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof arecombinant PHI-4 polypeptide. In some embodiments methods are providedfor protecting a plant from an insect pest, comprising expressing in theplant or cell thereof a recombinant pesticidal protein of SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NOs: 51-819 or variants thereof.

Insect Resistance Management (IRM) Strategies

Expression of B. thuringiensis δ-endotoxins in transgenic corn plantshas proven to be an effective means of controlling agriculturallyimportant insect pests (Perlak, et al., 1990; 1993). However, insectshave evolved that are resistant to B. thuringiensis δ-endotoxinsexpressed in transgenic plants. Such resistance, should it becomewidespread, would clearly limit the commercial value of germplasmcontaining genes encoding such B. thuringiensis δ-endotoxins.

One way to increasing the effectiveness of the transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests is to use provide non-transgenic (i.e.,non-insecticidal protein) refuges (a section of non-insecticidalcrops/corn) for use with transgenic crops producing a singleinsecticidal protein active against target pests. The United StatesEnvironmental Protection Agency(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which canbe accessed using the www prefix) publishes the requirements for usewith transgenic crops producing a single Bt protein active againsttarget pests. In addition, the National Corn Growers Association, ontheir website:(ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can beaccessed using the www prefix) also provides similar guidance regardingrefuge requirements. Due to losses to insects within the refuge area,larger refuges may reduce overall yield.

Another way of increasing the effectiveness of the transgenicinsecticides against target pests and contemporaneously reducing thedevelopment of insecticide-resistant pests would be to have a repositoryof insecticidal genes that are effective against groups of insect pestsand which manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic tothe same insect species, each insecticide being expressed at efficaciouslevels would be another way to achieve control of the development ofresistance. This is based on the principle that evolution of resistanceagainst two separate modes of action is far more unlikely than only one.Roush for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998)353:777-1786). Stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. The U.S.Environmental Protection Agency requires significantly less (generally5%) structured refuge of non-Bt corn be planted than for single traitproducts (generally 20%). There are various ways of providing the IRMeffects of a refuge, including various geometric planting patterns inthe fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments the PHI-4 polypeptides of the disclosure are usefulas an insect resistance management strategy in combination (i.e.,pyramided) with other pesticidal proteins include but are not limited toBt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins,and the like.

Provided are methods of controlling Lepidoptera and/or Hemiptera insectinfestation(s) in a transgenic plant that promote insect resistancemanagement, comprising expressing in the plant at least two differentinsecticidal proteins having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orHemiptera insect infestation in a transgenic plant and promoting insectresistance management the at least one of the insecticidal proteinscomprise a PHI-4 polypeptide insecticidal to insects in the orderColeoptera.

In some embodiments the methods of controlling Lepidoptera and/orHemiptera insect infestation in a transgenic plant and promoting insectresistance management the at least one of the insecticidal proteinscomprises a protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 51-819 or variants thereof, insecticidal to insects in the orderColeoptera.

In some embodiments the methods of controlling Lepidoptera and/orHemiptera insect infestation in a transgenic plant and promoting insectresistance management comprise expressing in the transgenic plant aPHI-4 polypeptide and a cry protein insecticidal to insects in the orderColeoptera having different modes of action.

In some embodiments the methods of controlling Coleoptera insectinfestation in a transgenic plant and promoting insect resistancemanagement comprise in the transgenic plant a protein of SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 51-819 or variants thereof and acry protein insecticidal to insects in the order Lepidoptera and/orHemiptera having different modes of action.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Hemiptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of a PHI-4 polypeptide insecticidal tothe insect species in combination with a second insecticidal protein tothe insect species having different modes of action.

Also provided are methods of reducing likelihood of emergence ofColeoptera insect resistance to transgenic plants expressing in theplants insecticidal proteins to control the insect species, comprisingexpression of a protein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 51-819 or variants thereof, insecticidal to the insect species incombination with a second insecticidal protein to the insect specieshaving different modes of action.

Also provided are means for effective Coleoptera insect resistancemanagement of transgenic plants, comprising co-expressing at high levelsin the plants two or more insecticidal proteins toxic to Lepidopteraand/or Hemiptera insects but each exhibiting a different mode ofeffectuating its inhibiting growth or killing activity, wherein the twoor more insecticidal proteins comprise a PHI-4 polypeptide and a cryprotein. Also provided are means for effective Lepidoptera and/orHemiptera insect resistance management of transgenic plants, comprisingco-expressing at high levels in the plants two or more insecticidalproteins toxic to Lepidoptera and/or Hemiptera insects but eachexhibiting a different mode of effectuating its inhibiting growth oractivity, wherein the two or more insecticidal proteins comprise aprotein of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 51-819or variants thereof and a cry protein.

In addition, methods are provided for obtaining regulatory approval forplanting or commercialization of plants expressing proteins insecticidalto insects in the order Coleoptera, comprising the step of referring to,submitting or relying on insect assay binding data showing that thePHI-4 polypeptide does not compete with binding sites for cry proteinsin such insects. In addition, methods are provided for obtainingregulatory approval for planting or commercialization of plantsexpressing proteins insecticidal to insects in the order Coleoptera,comprising the step of referring to, submitting or relying on insectassay binding data showing that the protein of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 51-819 or variant thereof does not competewith binding sites for cry proteins in such insects.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which thepolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a lepidopteran, coleopteran,dipteran, hemipteran or nematode pest, and the field is infested with alepidopteran, hemipteran, coleopteran, dipteran or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. By “biomass” is intended anymeasured plant product. An increase in biomass production is anyimprovement in the yield of the measured plant product. Increasing plantyield has several commercial applications. For example, increasing plantleaf biomass may increase the yield of leafy vegetables for human oranimal consumption. Additionally, increasing leaf biomass can be used toincrease production of plant-derived pharmaceutical or industrialproducts. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing a PHI-4 polypeptide disclosedherein. Expression of the PHI-4 polypeptide results in a reduced abilityof a pest to infest or feed on the plant, thus improving plant yield.

These and other changes may be made to the invention in light of theabove detailed description. In general, in the following claims, theterms used should not be construed to limit the invention to thespecific embodiments disclosed in the specification and the claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books, or otherdisclosures) in the Background of the Invention, Detailed Description,and Examples is herein incorporated by reference in their entireties.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight;temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Experimentals Example 1: Generating PHI-4 Genes

Polynucleotides having single codon substitutions compared to the PHI-4polypeptide of SEQ ID NO: 1 were generated. As described in the examplesbelow, the corresponding PHI-4 polypeptides were expressed, purified andassayed for WCRW insecticidal activity in order to assess thecorresponding activity diversity. A reverse mutagenesis primer and acomplementary forward mutagenesis primer were designed to create thedesired amino acid substitution(s) at the site(s) of interest. Typicallythe mutagenesis primer was between 30 to 45 bases in length with two ormore bases, usually 10 to 15, on both sides of the site of interest. Inthe case of saturation mutagenesis, degenerated primers that cover allpossible amino acid residues were used. Unless otherwise noted, themutagenic reactions were carried out using Agilent's QuikChange™Lightening Site-Directed Mutagenesis kit. Materials provided in the kitare QuikChange™ Lightening Enzyme, 10× QuikChange™ Lightning Buffer,dNTP mix, QuikSolution reagent and Dpn1 restriction enzyme according tothe manufactures directions.

PCR amplifications were typically carried out with Expand™ High FidelityPCR system (Roche, Switzerland) in 50 ul containing 50-100 ng templates,0.4-2 μM primer pair, 200 μM dNTPs and 2 Units of DNA polymerase. Themutagenesis reaction was initiated by pre-heating the reaction mixtureto 94° C. for 3 min, followed by 16 cycles of the following cyclingprogram: 94° C. for 1 min, 52° C. for 1 min and 68° C. for 8, 12, 16 or24 min according to the length of template. The mutagenesis reaction wascompleted by incubation at 68° C. for 1 h. The PCR-amplificationproducts were evaluated by agarose gel electrophoresis. The PCR productswere purified by QIAquick™ PCR purification kit (Qiagen, Germany) andfurther treated with the restriction enzyme Dpn1. An aliquot of 1 μl ofthis PCR product was typically transformed into BL21(DE3) cells andinoculated on Luria-Bertani (LB) plate containing 100 μg/ml ampicillin.About 10 colonies in the case of single amino acid mutation or 48 ormore colonies for saturation mutagenesis were selected and plasmid DNAwas isolated for sequencing. Two step sequencing was used, first forspecific mutation site(s) with one sequencing primer followed by fulllength sequence confirmation with multiple sequencing primers.

Example 2: Purification of MBP::PHI-4 Fusion Polypeptides

Polynucleotides encoding PHI-4 polypeptides were expressed in a modifiedpMAL vector (New England Bio Lab) as a fusion (i.e. MBP::PHI-4; SEQ IDNO: 6) with MBP (maltose binding protein). The pMAL vector was modifiedto attach a 6× His tag to the N-terminus of MBP. In order to clone thepolynucleotide encoding the MBP::PHI-4 fusion protein (SEQ ID NO: 6),Sph1 and BamH1 sites were engineered in the vector at the cloning site.The polynucleotide (SEQ ID NO: 1) encoding the PHI-4 polypeptide (SEQ IDNO: 2) was amplified with a forward primer (SEQ ID NO: 32) overlappingthe Sph1 site and a reverse primer (SEQ ID NO: 33) overlapping the BamH1site. This PCR product was digested with Sph1 and BamH1 and cloned intopMAL that was precut with the same enzymes. The forward primer wasdesigned such that polynucleotides encoding both MBP and PHI-4polypeptide (SEQ ID NO: 2) within the MBP::PHI-4 gene (SEQ ID NO:5) wereligated in frame. The plasmid containing the polynucleotide (SEQ ID NO:5) encoding the MBP::PHI-4 polypeptide (SEQ ID NO: 6) was transformedinto E. coli BL21(DE3) cells. The BL21(DE3) cells were grown inMagicMedia™ (Life Technologies) in either 96 deep well plates or flasksin a shaker running at 250 rpm at 37° C. for 8 hrs. followed by 16° C.for 48-60 hrs. During the 16° C. incubation, the MBP::PHI-4 polypeptidefusion protein accumulated in the BL21 (DE3) cells as a soluble protein.

In order to purify the MBP::PHI-4 fusion protein (SEQ ID NO: 6), the E.coli cells were harvested by centrifugation and treated in a lysozymesolution consisting of 2 mg/ml lysozyme in 50 ml sodium phosphate bufferat pH 8.0 containing 300 mM NaCl, 2 U/ml endonuclease (Epicentre) and 5mM MgCl₂ for 3 hrs. at 37° C. with gentle shaking. The lysozyme treatedE. coli cells were then disrupted with 1% Triton X100 and clear lysatecontaining the MBP::PHI-4 proteins were prepared by centrifugation at4000 rpm, 30 min (96 well plates) or 9000 rpm (flask produced samples).His tagged MBP-PHI-4 polypeptide fusion proteins were purified from theclear lysates by affinity chromatography using NiNTA agarose (catalog#30450; Qiagen) following the manufacturer's standard procedure. Forhigh throughput protein purification, Pall 96 deep well filter plates(Pall Corporation; Catalogue #5051) were used for the affinitychromatography. The purified MPB::PHI-4 polypeptide fusion protein waseluted from NiNTA agarose and passed through Sephadex G25 to change thephosphate buffer to 25 mM HEPES-NaOH, pH 8 and used in insect bioassaysfor determining the insecticidal activity against Western Corn Rootworm(WCRW). Calipar GXII capillary electrophoresis with a protein chip(Agilent; catalogue #P/N760499) was used to determine the MPB::PHI-4polypeptide concentrations. The protein analysis was repeated at least 3times until the final concentration was within the predetermineddeviation (less than 10%). Unless otherwise noted, the PHI-4polypeptides disclosed herein were expressed, purified and assayed forWCRW insecticidal activity as maltose binding protein fusions (i.e.MBP::PHI-4; SEQ ID NO: 6) as described above.

Example 3: Determination of WCRW Insecticidal Activity of MBP::PHI-4(SEQ ID NO: 6) and MBP::PHI-4-SFR12-004 (SEQ ID NO: 31) Polypeptides

The activity of MBP::PHI-4 (SEQ ID NO: 6) and MBP::PHI-4-SFR12-004 (SEQID NO: 31; Example 8) polypeptides against WCRW (western corn rootworm,Diabrotica virgifera virgifera) was determined in an artificial dietfeeding assay essentially as described by Cong, R. et al. (Proceedingsof the 4th Pacific Rim Conferences on Biotechnology of Bacillusthuringiensis and its environmental impact, pp. 118-123, ed. by R. J.Akhurst, C. E. Beard and P. Hughes, published in 2002, Canberra,Australia). The assays were conducted on an artificial diet containingdilutions of these polypeptides. The MBP::PHI-4-SFR12-004 polypeptidefusion (SEQ ID NO:31) and MBP::PHI-4 (SEQ ID NO: 6) fusions wereprepared as above, and 10 μL of protein samples were mixed with 50 μL ofmolten (40-50° C.) artificial insect diet especially prepared forDiabrotica sp. with low temperature melting agarose, whey protein andwheat germ. The diet-PHI-4 polypeptide mixture was placed in each wellof a 96 well micro-titer plate. Four or more neonate WCRW larvae wereplaced in each well to feed for 4 days at 25° C. and the response ofinsects towards the proteins was scored using a 0-3 numerical scoringsystem based on the size and mortality of the larvae in each well. If noresponse (or normal growth) was seen, a score of 0 was given. When thegrowth was slightly retarded, a score of 1 was given. A score of 2 meantthat the larvae were severely retarded in growth (close to neonatesize). A score of 3 meant death to all the larvae in the well. Thepercent response (% Response) for each treatment was calculated bydividing the total score, a sum of scores from replicated wells for eachtreatment, by the total highest possible scores and multiplying by 100to yield “% Response”. For example, if one treatment (one sample, onedose) had 6 replicated wells, the total highest possible score would be3×6=18. An observed set of scores of 3, 2, 2, 3, 2, 2 for six wells at agiven dose for a given variant would result in (14/18)×100=78% Response.

Fast Activity Evaluation (FAE) Analysis:

The PHI-4 polypeptides at two concentrations were assayed along with 4doses (two-fold dilutions bracketing the EC50) of the referenceMBP::PHI-4 fusion protein (SEQ ID NO: 6) within one 96-well assay plate.The concentrations of the PHI-4 polypeptides were within the 4 doses ofthe reference protein concentrations, preferably around the middle pointof the 4 dose concentrations. Each sample plate contained the referenceMBP::PHI-4 protein (SEQ ID NO: 6) in a significant number of wells suchas 16 wells in 4 separate doses. In each plate, up to 80 MBP::PHI-4polypeptide variants were included and assayed for activity comparisonwith the reference PHI-4 polypeptide protein. From a sample plate, 10 ulof samples from each well were picked by multi-channel pipette anddispensed in one assay plate containing 50 ul molten diet in each welland mixed on a shaker. This process of producing the assay plate wasrepeated as many as 6 times or more to produce a desired number ofreplicate assay plates. After the diet was solidified and cooled to 4°C., neonate WCRW larvae were placed in each well, the plate was sealedwith perforated Mylar film and incubated in a constant temperatureincubator at 25° C. After 4 days, the insect responses were scored undera magnifying glass. The sigmoid dose-response values (Responses) wereconverted to linear probit dose-response values using SAS-JMP®,Generalized Linear Model, Binomial Response, Probit). The response foreach protein in replicates was summed, this sum was compared with theprobit dose-response line of the activity reference protein and thenominal fold improvement in potency was calculated. This nominal foldimprovement estimated for a given dose in a given experiment is definedas the Fast Activity Evaluation Guide Number (FAEGN). For example, if aPHI-4 polypeptide showed a certain % response value at 40 ppm andcomparison to the Probit curve indicated that the same response ispredicted for the reference protein at 100 ppm, then the FAE GuideNumber is 2.5 (100/40). According to this analysis, the PHI-4polypeptide is nominally 2.5 times more potent than the reference PHI-4polypeptide protein. The FAE assay was typically done with 2 differentdoses of PHI-4 polypeptides at a time and performed in three independentexperiments, generating 6 FAEGN for each mutant in a typical FAEevaluation. The mean FAEGN is calculated to yield the Mean FAE Index(MFI). As used herein “Mean FAE Index” (MFI) refers to the mean ofmultiple FAEGN (typically 6 or more); unless otherwise indicated MFI isunderstood to be an arithmetic mean of FAEGN. For each protein, a twosided t-test was done comparing the multiple FAEGN from the clone withFAEGN values from the reference protein (typically 48-96 FAEGN of SEQ IDNO: 6). The two-sided t-test was done between these 48-96 FAEGNassociated with the reference protein and the 6 or more FAEGN associatedwith the variant of interest. The Bonferroni correction was used toevaluate p-values (number of novel proteins/alpha) for the hypothesisthat a given variant is significantly different in insecticidal activitycompared to the reference protein, MBP::PHI-4 fusion protein (SEQ ID NO:6) unless otherwise noted. The Bonferroni correction was used toevaluate p-values (number of novel proteins/alpha) for the hypothesisthat a given variant is significantly different in insecticidal activitycompared to the reference protein, MBP::PHI-4 fusion protein (SEQ ID NO:6) unless otherwise noted.

Effective Concentration₅₀ (EC50) evaluation: Variants of particularinterest were assayed at higher power in more extensive dose responsecurves to more accurately estimate the EC50s. The preparation of doseresponse, infestation, incubation and scoring were as described for theFAE assay format. EC50 determinations were typically carried out with ano insecticidal protein control plus seven two-fold dilutions bracketingthe expected EC50 (typically 100 ppm for MBP::PHI-4; SEQ ID NO: 6) and24 or more replicate wells within a given experiment. As used herein,the EC50 is defined as the predicted point with 50% response in thescoring scheme. It is a combination of growth or feeding Inhibition andlethal responses. In order to determine EC50 values, each treatment (onedose) was repeated 6 or more, usually 24, times.

Data from exemplary FAE and EC50 determinations are given in FIGS. 1 and2 respectively. MBP::PHI-4-SFR12-004 (SEQ ID NO: 31; Example 8) is avariant with improved insecticidal activity. FIG. 1 shows the primarydata for a typical FAE assay. Proteins were purified and quantified asdescribed above. The % response in a typical FAE assay is given on the yaxis. The protein concentration (toxin portion of the protein only) isgiven on the x axis. The geometric mean FAE Index in this instance is4.0. FIG. 2 shows the data for a typical EC50 measurement. Proteins werepurified and quantified as described above. The fractional response(multiply by 100 to get “% Response”) is given on the y axis. Theinferred EC50s are 245 ppm (MBP::PHI-1 of SEQ ID NO: 6) and 48 ppm(SFR12-004; SEQ ID NO: 31). The data indicate that the insecticidalactivity of MBP::PHI-4-SFR12-004 (SEQ ID NO: 31) is increased relativeto that of MBP::PHI-4 (SEQ ID NO: 6).

The MBP::PHI-4 fusion is rapidly cleaved in the presence of insect gutfluid to yield MBP and mature PHI-4 protein and it is believed that theinsecticidal activity is due to the cleaved toxin molecules. TheMBP::PHI-4 fusion protein (SEQ ID NO:6) was digested with 1/100 (w/w)Factor Xa (New England Biolabs) at 25° C. overnight and the PHI-4polypeptide was purified by Superdex 200 column chromatography utilizingthe size difference and a weak affinity of MBP to Superdex. TheMPB::PHI-4 fusion was also cleaved with trypsin and the mature PHI-4polypeptide (derived from SEQ ID NO: 6) was purified. The mature,purified PHI-4 polypeptide derived from Factor Xa or trypsin cleavagehas a MW of ˜60 kDa as measured on SDS-PAGE gels and is fully reactiveto polyclonal antisera that also react with the MBP::PHI-4 fusionprotein. The EC50 of this “mature” PHI-4 polypeptide fragment is withinexperimental error of the EC50 of the MBP::PHI-4 parental protein (SEQID NO: 6, as calculated on the basis of ppm associated with the toxinfragment and excluding ppm associated with the MBP domain of SEQ ID NO:6.

Example 4: Single Amino Acid PHI-4 Polypeptide Variants (S1-S4)

A set of 209 PHI-4 polypeptide single amino acid substitution variantsspread across both the N and C terminal portions of the protein weremade and characterized (Megatable 1; MUT ID: 1-209). The mutagenesistemplate was the polynucleotide of SEQ ID NO: 5 encoding PHI-4 (SEQ IDNO: 6) as an MBP fusion. The mutations were made using the QuickChangekit (Agilent; Catalogue #200524) essentially as described in Example 1.The particular amino acid substitutions relative to PHI-4 (SEQ ID NO: 2)are as indicated in Megatable 1. For example in Megatable 1, the PHI-4polypeptide identified as MUT ID: 1 has a valine substituted for thenative amino acid alanine at position 202 of PHI-4 (SEQ ID NO: 2) andreferred to “A202V”. The polypeptide variant for which activity isreported was prepared as an MBP fusion that is identical to SEQ ID NO: 6except for this single amino acid substitution. In a similar manner, MUTID: 1-872 (Megatable 1) are all made in the context of SEQ ID NO: 6; MUTID: 873-910 (Megatable 1) are made in the context of SEQ ID NO: 8; MUTID: 911-1135 (Megatable 1) are made in the context of SEQ ID NO: 10. Allpolypeptides of Megatable 1 were expressed and purified as MBP fusionsas described in Example 2. The PHI-4 polypeptides were expressed as MBPfusions and purified as described in Example 2. The assay protocol forWCRW insecticidal activity of the PHI-4 polypeptides was essentially asdescribed for FAE assays in Example 3 using SEQ ID NO: 6 as thereference protein. For the analysis of the data a “Mean Deviation Score”was calculated rather than a mean FAE Index. This is a related metricthat is calculated as follows. Data from four two-fold dilutions ofMBP::PHI-4 (typically about 25, 50, 100 and 200 ppm final concentrationof PHI-4 fragment in artificial insect diet) is plotted on a Probitplot. The difference between response expected for MBP::PHI-4 (SEQ IDNO: 6) at a given dose, based on the Probit curve, and that observed forthe mean score for a PHI-4 polypeptide variant at that given dose iscalculated and this difference is defined as the Deviation Score. Anegative Deviation Score indicates that the response is lower than thatwhich is expected for the relevant parental backbone (typicallyMBP::PHI-4; SEQ ID NO: 6) at the same concentration and indicates thatthe variant is nominally less active than the parental backbone. Apositive Deviation Score indicates that the variant is nominally morepotent than the parental backbone. As used herein, the Mean DeviationScore refers to the arithmetic mean of multiple Deviation Scores whichare typically derived from independent experiments. The Mean DeviationScore is used to estimate rank order of activities associated with a setof variants within a given experiment. The Mean Deviation Scores for the209 variants of this example are given in Megatable 1 and is typicallyan average of at least three independent Deviation Score measurements.

Example 5: Single Amino Acid Substitution Mutants #2 (SFR)

A BLAST search revealed that AXMI-205 is a bacterial perforin-likeprotein. Perforin proteins from Clavibactor michiganensis (GenBankAccession number: YP_001223127; SEQ ID NO: 49), Laccaria bicolor(GenBank Accession number:XP_001885969; SEQ ID NO: 48), Marinomonas sp.(GenBank Accession number: ZP_01077945; SEQ ID NO: 38), Nematostellavectensis (GenBank Accession number: XP_001617993; SEQ ID NO: 50),Photorhabdus luminescens (GenBank Accession number: NP_928713; SEQ IDNO: 821), and Serratia proteamaculans (GenBank Accession number:YP_00147861; SEQ ID NO: 820) were found to be homologous to AXMI-205.The N-terminal region of up to 311 amino acid of AXMI-205 (SEQ ID NO:35) is highly homologous to those perforin proteins. Among thoseperforin proteins, the 3D X-ray structure of Photorhabdus perforin-likeprotein has been published (Science 317, 1548-1551, 2007; PDB ID: 2QP2;SEQ ID NO: 822). According to the leading theory of perforin mode ofaction, the protein inserts 5 alpha helices of its N-terminal regioninto the target host membrane to form a large size pore (Proc. Natl.Acad. Sci. USA 102, 600-605, 2005; Immunology Today, 16, 194-201, 1995).In the Photorhabdus 2QP2 structure, there are two loops between Alpha Cand D and Alpha I and J are considered to be the site for initiating themembrane insertion of those 5 alpha helices. These loops are calledmembrane insertion initiation loops. The primary sequences of thoseperforin proteins listed below were aligned using Vector NTI Align Xfunction in order to identify the membrane insertion loop sequences ofAXMI-205 (SEQ ID NO: 35). Amino acid sequences of V92-A103 and G211-E220of SEQ ID NO: 6 were aligned with the membrane insertion initiationloops identified in the 2QP2 structure. A number of acidic, basic andother hydrophilic amino acids were found in the AXMI-205 (SEQ ID NO: 35)membrane insertion loops indicating these loops are exposed to thesolvent.

Based on this homology model and prior mutation-activity relationshipdata, another 664 single amino acid substitution PHI-4 polypeptidevariants were made in order to assess the sequence-insecticidalactivity-relationships at the selected positions. This set of 664 pointmutations (Megatable 1; MUT ID: 210-872) were made with MBP::PHI-1polypeptide of SEQ ID NO 5 as the DNA template. Mutations were madeaccording to the method of Example 1. The mutagenesis oligonucleotidesused to create an exemplary mutant (PHI-4-R97D; SEQ ID NO: 7) are SEQ IDNO: 13 and SEQ ID NO: 14. The other PHI-4 polypeptides (Megatable 1; MUTID: 210-872) were made in the same manner using mutagenesisoligonucleotides designed to create the selected substitutions at thedesired residues of the protein. The resulting PHI-4 polypeptides werepurified as MBP fusions and the activity measured in the FAE assayformat or EC50 assay format as described in Examples 2 & 3. The Mean FAEIndices associated with these 664 PHI-4 polypeptides are given in lines210-872 of Megatable 1.

Example 6: SFR16 Point Mutants

Another set of 38 single amino acid substitution PHI-4 polypeptidevariants (Megatable 1; MUT ID: 873-910) were made with MBP::PHI-4-R97D(SEQ ID NO 7; Example 5) as the backbone. The substitutions wereselected based on the sequence-activity relationships inferred from thePHI-4 polypeptide variants of the preceding Examples. Mutations weremade according to the method of Example 1. Variants were purified as MBPfusions and activity measured in FAE assay format as described inExamples 2 & 3. The Mean FAE Indices associated with these mutants aregiven in lines 873-910 of Megatable 1. The reference for the Mean FAEIndex in this example is SEQ ID NO: 8.

Example 7: PSR3 Mutants

The C-terminal domain of AXMI-205 shares significant homology withproteins that have β-prism 3D structural folding patterns. In thetypical β-prism, the oligosaccharide binding site is almost always in acavity formed between the apex and belt-loops from the same Greek-keymotif. The primary amino-acid sequence motif has been identified asG-X₃-D (SEQ ID NO: 39) in banana lectin, which has two binding sites(Meagher J L et al Glycobiology 15:1033-42; 2005). The primary sequenceof the PHI-4 polypeptide of SEQ ID NO: 2 contains 3 such motifs. Thecanonical structure of these motifs is indicated in FIG. 3. In addition,the G-X₃-D motif (SEQ ID NO: 39) can be extended toward the N-terminaldirection into the D-X-G-[S/T]-G-X₃-D motif (SEQ ID NO: 40), which arepresent 1, 2 or 3 times in 24 proteins that are orthologous to theC-terminal portion of AXMI-205 (GenBank accession numbers: gi|136474758;gi|136444345; gi|136141087; gi|143658948; gi|142085802; gi|135275135;gi|138446054; gi|294814724; gi|170109524; gi|156316804; gi|156377786;gi|170109526; gi|77456557; gi|1209377; gi|302823768; gi|302532087;gi|256764986; gi|302787479; gi|302823738; gi|169762636; gi|302766657;gi|270056485; gi|302792467; gi|238488445;). Each of the 3 loops in thePHI-4 polypeptide of SEQ ID NO: 2 has potential to bind anoligosaccharide, a putative binding receptor present in WCRW mid-gutcell membrane surface.

As indicated in Megatable 1, 225 PHI-4 polypeptide variants (MUT IDs:911-1135) were made to introduce an additional amino acid substitutioninto the PHI-4 polypeptide of PHI-4-D09 (SEQ ID NO: 10) usingMBP::PHI-4-D09 (SEQ ID NO: 9) as the DNA template. The PHI-4-D09backbone contains the following substitutions relative to SEQ ID NO: 2:L401, Y98F, L145V, L163V, 1172L, V3551, and P412A (numbers are relativeto the PHI-4 polypeptide backbone of SEQ ID NO: 2. Mutagenesis was doneby a modification of the method of Dominy et al (Methods in MolecularBiology, Vol. 235:209-223; 2003). Briefly, “NNK” mutagenesis at position396 was done as follows. A pMAL vector encoding SEQ ID NO: 9 wasamplified by inverse PCR for 20 cycles using SEQ ID NO: 15 & SEQ ID NO:16. The PCR product was diluted 10-fold, subjected to one additionalround of amplification using SEQ ID NO: 16 & SEQ ID NO: 17. The PCRproduct was purified on QuiaQuick™ column, phosphorylated with T4polynucleotide kinase, circularized with T4 DNA ligase and transformedinto E. coli BL21(DE3) cells. Candidate colonies were amplified bycolony PCR and the PCR product was sequenced first with a single primerto confirm the presence of the desired mutation and subsequentlysequenced fully with multiple primers to identify clones with noadditional mutations. All other PHI-4 polypeptides of this example(Megatable 1, lines 911-1135) were made by a similar manner usingmutagenesis oligonucleotides designed to create the selectedsubstitutions at the desired residues of the protein. Positions withmultiple desired mutations were made with degenerate forward primerswhereas positions with only one desired mutation were made withnon-degenerate primers. Clones with the desired sequences were used toexpress protein essentially as described in Example 2. Proteinpurification, activity measurements and statistical analysis was doneessentially as described in example 3. The Mean FAE Index reflects thefold difference relative to PHI-4 polypeptide of SEQ ID NO: 6. The MeanEC50 of PHI-4-D09 (SEQ ID NO: 10) was measured at high statistical powerand is 1.3-fold improved relative to MBP::PHI-4 (SEQ ID NO: 6). PHI-4polypeptides of this example with Mean FAE Index >1.3× are deemednominally improved relative to the parental backbone (MBP::PHI-4-D09;SEQ ID NO: 10) and diversity meeting this criterion was used forproduction of subsequent combinatorial mutants.

Example 8: Identification of Combinatorial Mutants of PHI-4 Polypeptideswith Improved Insecticidal Activity as Measured in an Artificial InsectDiet Feeding Assay

Activity diversity identified in Examples 4-7 was used to create 192combinatorial PHI-4 polypeptide variants (SEQ ID NO: 51-242; Megatable2). The PHI-4 polypeptide variants were made by sequential pointmutagenesis by the method of Example 1. In all cases, the indicatedPHI-4 polypeptide variants were made as MBP fusions with the same linkeras is indicated in SEQ ID NO 6. The MBP::PHI-4 polypeptides wereexpressed and purified as indicated in Example 2 & 3. PurifiedMBP::PHI-4 polypeptides were assayed in FAE assays to derive a Mean FAEIndex or in EC50 assays as indicated in Example 3. Thirty-sevenexemplary active PHI-4 polypeptide variants with increased Mean FAEIndices are given in Table 3, along with the sequence variation relativeto SEQ ID NO: 6. The substitutions relative to PHI-4 polypeptide of SEQID NO: 2 are given in the right-most column. All proteins were expressedand purified as MBP fusions proteins. The reference protein for the MeanFAE Index is MPB::PHI-4 (SEQ ID NO: 6). The functional data on all ofthe PHI-4 polypeptides of this example is given in lines 51-242 ofMegatable 2.

TABLE 3 SEQ Ex. ID Mutation List # NO: Alias FAE p value (vs SEQ ID NO:2) 8 148 SFR11- 27.4 1.36E−13 R097D, K099L, E220D, 001 K289L, R293Q 8225 SFR17- 20.9 0.001577 D042N, E046N, R097D, 013 K099L, E220D, K289L,R293Q, S333K, G336A, S401H, K402H 8 226 SFR17- 20.1 1.25E−07 D042N,E046N, R097D, 019 K099L, E220D, K289L, R293Q, S333K, G336A, V355I,S401H, K402H, P412A 8 227 SFR17- 19.5 1.48E−09 D042N, E046N, R097D, 014K099L, E220D, K289L, S333K, G336A, S401H, K402H, P412A 8 228 SFR17- 19.05.42E−09 D042N, E046N, R097D, 011 K099L, E220D, K289L, S333K, G336A,S401H, K402H 8 229 SFR17- 17.1 6.82E−10 R097D, K099L, E220D, 005 K289L,R293Q, S401H, K402H, P412A 8 73 SFR10- 17.1 1.48E−19 R097D, S333K, G336A032 8 230 SFR17- 16.5 7.08E−09 R097D, K099L, E220D, 018 K289L, R293Q,S333K, G336A, V355I, S401H, K402H, P412A 8 149 SFR11- 14.8 8.29E−15R097D, K099L 012 8 231 SFR17- 13.5 7.19E−16 D042N, E046N, R097D, 009K099L, E220D, K289L, R293Q, V355I, S401H, K402H, P412A 8 232 SFR17- 13.11.63E−10 D042N, E046N, R097D, 006 K099L, E220D, K289L, R293Q, S401H,K402H, P412A 8 233 SFR17- 12.9 5.18E−12 D042N, E046N, R097D, 016 K099L,E220D, K289L, R293Q, S333K, G336A, S401H, K402H, P412A 8 150 SFR11- 12.82.12E−10 R097D, K099L, E220D, 005 K289L 8 74 SFR10- 12.0 7.73E−11 R097D,S401H 042 8 234 SFR17- 11.7 1.82E−07 R097D, K099L, E220D, 012 K289L,R293Q, S333K, G336A, S401H, K402H 8 235 SFR17- 11.3 7.41E−06 D042N,E046N, R097D, 004 K099L, E220D, K289L, S401H, K402H, P412A 8 236 SFR17-10.5  2.1E−15 D042N, E046N, R097D, 017 K099L, E220D, K289L, S333K,G336A, V355I, S401H, K402H, P412A 8 151 SFR11- 9.9 2.08E−09 R097D, K289L014 8 237 SFR17- 9.7   1E−04 D042N, E046N, R097D, 003 K099L, E220D,K289L, R293Q, S401H, K402H 8 183 SFR13- 9.3   1.5E−11 R097D, K099L,E220D, 035 K289L, V355I, A396T, P412A, K520E 8 75 SFR10- 8.9 0.003487R097D, S333V, K520E, 72 Q527K 8 238 SFR17- 8.7 0.010038 D042N, E046N,R097D, 001 K099L, E220D, K289L, S401H, K402H 8 76 SFR10- 8.4 2.98E−05R097D, G462A, R464A, 056 K465M 8 77 SFR10- 8.2 1.37E−05 R097D, S333K,G336A, 036 E339N 8 239 SFR17- 8.2 4.75E−21 R097D, K099L, E220D, 015K289L, R293Q, S333K, G336A, S401H, K402H, P412A 8 152 SFR11- 8.21.76E−07 R097D, R293Q 015 8 153 SFR11- 8.1  1.7E−06 R097D, E220D, K289L010 8 240 SFR17- 7.6 5.14E−09 R097D, K099L, E220D, 002 K289L, R293Q,S401H, K402H 8 78 SFR10- 7.2  9.2E−12 R097D, S333V, G336A, 039 S338V 879 SFR10- 6.7 8.17E−06 R097D, S401H, K402H, 82 K520E, Q527K 8 80 SFR10-6.6 2.09E−14 R097D, S401H, K402H 045 8 81 SFR10- 6.4 2.15E−06 R097D,S401G, K402H, 87 K520E, Q527K 8 82 SFR10- 6.3 2.89E−08 R097D, G462A,R464K, 060 K465M 8 51 SFR5- 6.2 1.65E−05 R097D, R293Q, R416E, 014 K520E8 184 SFR13- 6.1 1.84E−13 R097D, K099L, E220D, 018 K289L, V355I, S401G,P412A, K520E 8 154 SFR11- 5.5 1.54E−09 R097D, E220D 013 8 241 SFR17- 5.55.11E−22 D042N, E046N, R097D, 007 K099L, E220D, K289L, V355I, S401H,K402H, P412A

Example 9: Identification of Combinatorial Mutants of PHI-4 Polypeptideswith Improved Insecticidal Activity as Measured in an Artificial WCRWInsect Diet Feeding Assay

Activity data from 315 PHI-4 polypeptide combinatorial variants (SEQ IDNOs 243-558) is provided in Megatable 2. Libraries were initiallyprepared by incorporation of diversity into SEQ ID NO: 5. The diversitywas largely derived from that described in Example 4. Oligonucleotidesencoding diversity at forty positions were incorporated into SEQ ID NO:5 in a DNA shuffling reaction essentially as described (Ness et al.,Nature Biotechnol. 20, 1251; 2002). Briefly, oligonucleotides typicallyof 30-45 bases in length encoding the diversity elements of interestwere mixed at 0.02-2 micromolar each in the presence of an appropriateconcentration of fragments of SEQ ID NO: 5. This reaction was assembled,rescued and cloned essentially as described for synthetic genes inExample 10 and as described (Ness et al., Nature Biotechnol. 20, 1251;2002). Improved variants from initial libraries were subjected to familyDNA shuffling essentially as described (A. Crameri, et al Nature 391,288; 1998). This family shuffled library was screened by methods similarto those described in Example 3 (FAE) and to those described in Example4 (Mean Deviation Score). Selected, improved PHI-4 polypeptide variantsfrom the second round of DNA shuffling were further diversified byrecombining N terminal and C terminal domains of elite clones using themethod of splicing by overlap extension (R. Horton, et al., Gene77:61-68; 1989) to yield novel variants. All variants were purified bythe method of Example 2 and assayed by the method of Mean Deviations ofExample 4. PHI-4 polypeptides variants identified are given in Megatable2 (SEQ ID NO: 243-558).

Example 10: Identification of Combinatorial Mutants of PHI-4Polypeptides with Improved Insecticidal Activity as Measured in anArtificial WCRW Insect Diet Feeding Assay

A set of 158 PHI-4 polypeptide combinatorial variants (SEQ ID NO:559-716) were prepared by total gene synthesis, essentially as describedby Stemmer et al (Gene 164:49-53; 1995). An additional treatment wasimplemented as described (Saaem, I. et al, Nucleic Acids Research,Published Nov. 29, 2011, 1-8). Briefly, in a typical gene synthesisreaction a set of oligonucleotides of 120 bases each encoding both topand bottom strands of the target gene were designed. Complementaryoligos typically overlap by 54-65 nucleotides. Oligos to make syntheticgenes are combined such that a final concentration of each oligo isapproximately 0.05-1 uM. Gene assembly is typically done with HerculaseII (Agilent) using the following cycling program: 98° C. 3 min followedby (96° C.×30 sec, 40° C.×30 sec, 72° C.×30 sec)×24 cycles. The initialPCR is then used as template for a second PCR in which a second pair ofprimers is used to amplify the fully synthetic gene product. Typical PCRconditions for the second synthetic gene reaction were 98° C. 3 minfollowed by (96° C. 30 sec, 50° C. 30 sec, 72° C. 35 sec), ×24 cycles.Reactions were analyzed by 1% E-gels (Invitrogen). A subsequenttreatment consisting of a re-annealing step, treatment with Cell(Transgenomics; Catalogue #706020) and subsequent amplification (25cycles) was done essentially as described (Saaem, I. et al, NucleicAcids Research, Published Nov. 29, 2011, 1-8). A third and finalamplification of the synthetic gene was done with similar PCR conditionsin a single cycle. The product of this reaction was purified and ligatedby Gibson ligation method (New England Biolabs; Catalogue ## E2611 L) toan appropriate vector transformed to chemical competent BL21(DE3) cells.Sequence verified clones (Lines 559-716 of Megatable 2) comprising thePHI-4 polypeptide were expressed as MBP fusions, purified and assayedessentially as described in Examples 2 and 3. Table 4 shows the SEQ IDNOs and substitutions relative to the PHI-4 polypeptide of SEQ ID NO: 2for twenty active variants. The mean FAE Index is calculated relative tothe MBP::PHI-4 backbone (SEQ ID NO: 6)

TABLE 4 SEQ Ex. ID MutationList # NO: Alias FAE p value (vs SEQ ID NO:2) 10 559 PSR1- 25.6 3.26E−05 D042N, Y098F, L145V, L153I, 1-076 I172L,Y206F, I283V, V355I, G359A, W389L, I410V, A417S 10 560 PSR1- 22.74.19E−05 D042N, Y098F, I283V, V355I 1-074 10 561 PSR1- 15.9 7.67E−06E046N, Y098F, L145V, Y171F, 2-145 I172L, D182Q, E278N, V355I, I410V,A417S, Q442E, V455I 10 562 PSR1- 11.6 1.46E−18 F043E, Y098F, L145V,Y171F, 2-082 I172L, Y206F, E278N, E339Q, V355I, V455I, W457N 10 563PSR1- 11.0 3.91E−05 F043E, Y098F, L145V, Y171F, 2-088 I172L, Y206F,E278N, V355I, Q442E, V455I, W457N 10 564 PSR1- 10.1 1.07E−10 D042N,F043E, Y098F, L145V, 2-094 Y171F, I172L, E278N, M354L, V355I, V455I,W457N 10 565 PSR1- 9.7  2.8E−08 D042N, F043E, Y098F, L145V, 2-110 Y171F,I172L, E278N, V355I, I410V, Q442E, V455I 10 566 PSR1- 8.5 0.003166D042N, Y098F, I283V, V355I, 1-073 A417S 10 567 PSR1- 7.2 1.03E−10 D042N,F043E, Y098F, L145V, 2-091 Y171F, I172L, V355I, Q442E, V455I, W457N 10568 PSR1- 7.0 8.39E−08 F043E, Y098F, L145V, Y171F, 2-149 I172L, Y206F,E278N, V355I, A417S, V455I, W457N 10 569 PSR1- 6.9 3.93E−10 D042N,F043E, Y098F, L145V, 2-087 Y171F, I172L, E278N, V355I, Q442E, V455I,W457N 10 570 PSR1- 6.9 1.01E−07 D042N, R097N, Y098F, L145V, 2-158 Y171F,I172L, V355I, 1410V, V455I 10 571 PSR1- 6.7 2.02E−05 E046N, Y098F,L145V, Y171F, 2-086 I172L, D182Q, E278N, V355I, Q442E, V455I, W457N 10572 PSR1- 6.5 0.010726 D042N, I052V, Y098F, L145V, 1-053 I172L, Y206F,I283V, V355I, H370R, I410V, P412A, A417S, T426S, T461S 10 573 PSR1- 6.25.28E−07 D042N, F043E, Y098F, L145V, 2-096 Y171F, I172L, V210I, I283V,M354L, V355I, V455I, W457N 10 574 PSR1- 6.1 2.92E−11 D042N, R097N,Y098F, L145V, 2-135 Y171F, I172L, V355I, H370R, Q442E, V455I 10 575PSR1- 5.9 0.018276 D042N, R097N, Y098F, L145V, 1-014 I172L, I283V,V355I, I410V, Q442E, V455I 10 576 PSR1- 5.8 5.02E−09 E046N, R097N,Y098F, L145V, 2-141 Y171F, I172L, V355I, Q442E, V455I 10 577 PSR1- 5.60.017252 E046N, Y098F, L145V, L163V, 1-006 I172L, Y206F, V210I, E339Q,V355I, A417S 10 578 PSR1- 5.3 7.35E−05 F043E, Y098F, L145V, Y171F, 2-095I172L, Y206F, E278N, M354L, V355I, V455I, W457N

Example 11: Identification of Combinatorial Mutants of PHI-4Polypeptides with Improved Insecticidal Activity as Measured in anArtificial Insect Diet Feeding Assay

Sixty-six PHI-4 polypeptide variants (SEQ ID NO: 717-783), containingpermutations of a number of substitutions, were made by total genesynthesis, essentially as described in Example 10. The substitutionswere done in the context of a backbone (PSR1-2-105; SEQ ID NO: 584 fromMegatable 2) containing the following substitutions relative to PHI-4polypeptide of SEQ ID NO: 2: E46N, R97N, Y98F, L145V, Y171F, 1172L,V3551, 1410V, V4551, and W457N. The resulting MBP::PHI-4 polypeptidefusion proteins were expressed, purified, assayed for insecticidalactivity on WCRW larvae and analyzed for insecticidal relative toMBP::PHI-4 (SEQ ID NO: 6) using the Mean FAE Index metric as describedin Examples 2 & 3. Table 5 shows the Mean FAE Indices, SEQ ID NOs andsubstitutions relative to PHI-4 polypeptide of SEQ ID NO: 2 for twentyactive PHI-4 polypeptide variants of this example. The Mean FAE Indexwas calculated relative to the MBP::PHI-4 backbone (SEQ ID NO: 6). Theinsecticidal activities of the PHI-4 polypeptides in Table 5 (Mean FAEIndex) reflect the arithmetic means of three independent experiments andare expressed as fold WCRW insecticidal activity improvement of thePHI-4 polypeptide variants relative to MBP::PHI-4 (SEQ ID NO: 6). Asindicated, the mean FAE Indices range from 0.26× to >8× (foldimprovement relative to MBP::PHI-4). The majority of the PHI-4polypeptides have increased insecticidal activity relative to MBP::PHI-4(FAE>1). The p values indicate that the measured differences relative toMBP::PHI-4 (SEQ ID NO: 6) are highly significant.

TABLE 5 Ex. SEQ ID Mutation List # NO: Alias FAE (vs SEQ ID NO: 2) 11717 PSR7- 10.0 E046N, R097N, Y098F, 141 L145V, Y171F, I172L, V355I,A396K, S401K, D403Y, I410V, V455I, W457N 11 718 PSR7-63 8.4 E046N,R097N, Y098F, L145V, Y171F, I172L, V355I, A396L, S401H, K402G, I410V,V455I, W457N 11 719 PSR7-89 8.1 E046N, R097N, Y098F, L145V, Y171F,I172L, V355I, A396L, S401H, D403Y, I410V, V455I, W457N 11 720 PSR7-946.6 E046N, R097N, Y098F, L145V, Y171F, I172L, V355I, A396G, S401K,I410V, V455I, W457N 11 721 PSR7- 6.1 E046N, R097N, Y098F, 106 L145V,Y171F, I172L, V355I, A396K, D403Y, I410V, V455I, W457N 11 722 PSR7-966.1 E046N, R097N, Y098F, L145V, Y171F, I172L, V355I, A396L, S401H,K402W, D403Y, I410V, V455I, W457N 11 723 PSR7- 5.0 E046N, R097N, Y098F,100 L145V, Y171F, I172L, V355I, A396K, K402H, I410V, V455I, W457N 11 724PSR7- 4.7 E046N, R097N, Y098F, 148 L145V, Y171F, I172L, V355I, A396G,S401K, K402G, D403Y, I410V, V455I, W457N 11 725 PSR7-98 4.7 E046N,R097N, Y098F, L145V, Y171F, I172L, V355I, A396K, S401H, K402W, D403Y,I410V, V455I, W457N 11 726 PSR7- 4.5 E046N, R097N, Y098F, 113 L145V,Y171F, I172L, V355I, A396K, S401K, I410V, V455I, W457N 11 727 PSR7- 4.5E046N, R097N, Y098F, 121 L145V, Y171F, I172L, V355I, S401H, I410V,V455I, W457N 11 728 PSR7-7 4.5 E046N, R097N, Y098F, L145V, Y171F, I172L,V355I, A396K, S401H, K402H, I410V, V455I, W457N 11 729 PSR7-86 4.2E046N, R097N, Y098F, L145V, Y171F, I172L, V355I, A396K, S401G, K402H,I410V, V455I, W457N 11 730 PSR7- 3.7 E046N, R097N, Y098F, 155 L145V,Y171F, I172L, V355I, S401H, K402G, D403Y, I410V, V455I, W457N 11 731PSR7- 3.6 E046N, R097N, Y098F, 116 L145V, Y171F, I172L, V355I, S401K,K402W, D403Y, I410V, V455I, W457N 11 732 PSR7-95 3.4 E046N, R097N,Y098F, L145V, Y171F, I172L, V355I, S401G, K402H, D403Y, I410V, V455I,W457N 11 733 PSR7-90 3.3 E046N, R097N, Y098F, L145V, Y171F, I172L,V355I, A396L, K402G, I410V, V455I, W457N 11 734 PSR7-97 3.3 E046N,R097N, Y098F, L145V, Y171F, I172L, V355I, A396G, K402H, I410V, V455I,W457N 11 735 PSR7-64 2.8 E046N, R097N, Y098F, L145V, Y171F, I172L,V355I, A396G, S401H, K402W, D403Y, I410V, V455I, W457N 11 736 PSR7-932.8 E046N, R097N, Y098F, L145V, Y171F, I172L, V355I, S401H, K402W,D403Y, I410V, V455I, W457N

Example 12: Identification of Combinatorial Mutants of PHI-4Polypeptides with Improved Insecticidal Activity as Measured in anArtificial WCRW Insect Diet Feeding Assay

Thirty six PHI-4 polypeptide variants (Megatable 2; SEQ ID NO: 784-819),containing permutations of eleven substitutions (E220D, G336A, K099L,K289L, K402H, K520E, P412A, R097D, S333K, S401H, V3551; numbering schemeas per SEQ ID NO:2; all variants made as MBP fusions as indicated inMegatable 2), were made using site directed mutagenesis as described inExample 1 and PHI-4 polypeptide variants were expressed, purified,assayed for insecticidal activity on WCRW larvae and analyzed forinsecticidal activity relative to PHI-4 polypeptide of SEQ ID NO: 2 asdescribed in Examples 2 & 3. Table 6 shows the Mean FAE Indices, SEQ IDNOs and substitutions relative to the PHI-4 polypeptide of SEQ ID NO: 2for twenty active PHI-4 polypeptide variants of this example. The meanFAE Index is calculated relative to the MBP::PHI-4 backbone (SEQ ID NO:6). The mean FAE indices reflect the arithmetic means of threeindependent experiments. As indicated, the mean FAE indices range from4.5 to >8. The p values indicate that the measured differences relativeto MBP::PHI-4 (SEQ ID NO: 6) are highly significant.

TABLE 6 SEQ ID Ex. # NO: Alias FAE MutationList (vs SEQ ID NO: 2) 12 784SFR15- 18.8 R097D, K099L, E220D, K289L, S333K, 009 G336A, S401H, K402H,K520E 12 785 SFR15- 8.0 R097D, K099L, E220D, K289L, S401H, 019 K402H,P412A 12 786 SFR15- 7.9 R097D, K099L, E220D, K289L, S333K, 021 G336A,S401H, K402H 12 787 SFR15- 7.9 R097D, K099L, E220D, K289L, S333K, 033V3551, S401H, K402H 12 788 SFR15- 7.3 R097D, K099L, E220D, K289L, V355I,020 S401H, K402H, P412A 12 789 SFR15- 7.0 R097D, K099L, E220D, K289L,S333K, 027 V355I, S401H 12 790 SFR15- 6.6 R097D, K099L, E220D, K289L,V355I, 036 K520E 12 791 SFR15- 6.6 R097D, K099L, E220D, K289L, S401H,007 K402H, P412A, K520E 12 792 SFR15- 5.5 R097D, K099L, E220D, K289L,S401H, 017 K402H 12 793 SFR15- 5.5 R097D, K099L, E220D, K289L, S333K,015 G336A, P412A 12 794 SFR15- 5.4 R097D, K099L, E220D, K289L, S401H,005 K402H, K520E 12 795 SFR15- 5.3 R097D, K099L, E220D, K289L, S333K,001 G336A, K520E 12 796 SFR15- 5.2 R097D, K099L, E220D, K289L, V355I 03012 797 SFR15- 5.1 R097D, K099L, S333K, G336A, S401H, 025 K402H, K520E 12798 SFR15- 5.0 R097D, K099L, E220D, K289L, S333K, 011 G336A, S401H,K402H, P412A, K520E 12 799 SFR15- 4.9 R097D, K099L, E220D, K289L, S333K,012 G336A, V355I, S401H, K402H, P412A, K520E 12 800 SFR15- 4.7 R097D,K099L, E220D, K289L, S333K, 029 V355I, S401H, P412A 12 801 SFR15- 4.7R097D, K099L, E220D, K289L, S333K, 016 G336A, V355I, P412A 12 802 SFR15-4.5 R097D, K099L, E220D, K289L, S333K, 010 G336A, V355I, S401H, K402H,K520E 12 803 SFR15- 4.5 R097D, K099L, E220D, K289L, S333K, 003 G336A,P412A, K520E

Example 13: Accordance Between FAE and EC50 Assays

Experimental data on Mean FAE Index and mean EC50 for twenty-five PHI-4polypeptide variants is given in FIG. 4. PHI-4 polypeptide variants werefirst tested in the FAE assay and then selected ones were retested in amultiple EC50 assays. In general, the fold improvement in mean FAE Indexis modestly larger than the fold improvement that was subsequentlymeasured in Mean EC50 measurements. This overall trend is as expectedfrom the phenomenon of regression toward the mean (International Journalof Epidemiology 2005; 34:215-220). All 25 PHI-4 polypeptides elected forretesting in EC50 assays repeated as being significantly improved. FIG.4 shows the EC50 measurements for representative variants from Megatable2 (SEQ ID NO: 610, SEQ ID NO: 595, SEQ ID NO: 584, SEQ ID NO: 591, SEQID NO: 576, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 79,SEQ ID NO: 81, SEQ ID NO: 150, SEQ ID NO: 150, SEQ ID NO: 149, SEQ IDNO: 167, SEQ ID NO: 167, SEQ ID NO: 164, SEQ ID NO: 164, SEQ ID NO: 170,SEQ ID NO: 170, SEQ ID NO: 795, SEQ ID NO: 794, SEQ ID NO: 784, SEQ IDNO: 799, SEQ ID NO: 785, SEQ ID NO: 788, SEQ ID NO: 786, SEQ ID NO: 796,SEQ ID NO: 787).

Example 14: Combinatorial Substitutions

Example 11 yielded numerous PHI-4 polypeptide variants with improvedinsecticidal activity based on combinatorial substitutions of the threelectin-like motifs described in Example 7. A combinatorial library wasprepared of 120 genes based on this diversity as follows. SEQ ID NOs 760& 761 each contains unique substitutions in loop 1. SEQ ID NOs 717-726;728-732; 734-737 & 760 contain unique substitutions in loop 2. SEQ IDNOs 761 and 758 contain unique substitutions in loop 3. Gene synthesiswas used to create a combinatorial library of these loop sequencesessentially as described in Example 10. These genes can be expressed andassayed for activity on WCRW larvae by the methods described above.

Example 15: Mutagenesis of Putative Protease Sensitive Sites of PHI-4Polypeptides

Trypsin was used to identify the site(s) where proteases possibly attack(Protease Accessible Sites) the PHI-4 polypeptide of SEQ ID NO: 2. ThePHI-4 polypeptide of SEQ ID NO: 2 in 50 mM Tris-HCl, pH8 was mixed with1/50 (weight/weight) trypsin and incubated for 1 hr at 37° C. It wasfound that protein was relatively resistant to trypsin, with noimmediate digestion down to the small fragments, but produced a 55 kDamajor band and 24 kDa minor band by SDS-PAGE analysis after theincubation. These two bands were excised from the gel and analyzed bymass spectrometry and N-terminal sequencing. The N-terminal sequencingrevealed SAANAGQLGN (amino acids 3-12 of SEQ ID NO: 2) for the 55 kDaprotein indicating that only two amino acid residues, Methionine andAlanine were missing from the N-terminal sequence. Themass-spectrometry, however, showed a loss of C-terminal sequence fromSer at 521 to Leu at 536 of SEQ ID NO: 2. This indicates that trypsindigested the PHI-4 polypeptide protein at the C-terminal side of Lys at520 of SEQ ID NO: 2. The N-terminal sequence of the 24 kDa band wasVDKVLLMD (amino acids 314 to 321 of SEQ ID NO: 2). The mass-spectrometryanalysis on the 24 kDa fragment confirmed the C-terminal region of PHI-4polypeptide of SEQ ID NO: 2 starting with Val at 314 relative to SEQ IDNO: 2 as shown by N-terminal sequencing and ending at Lys at 520relative to SEQ ID NO: 2. This indicated that trypsin digested the PHI-4polypeptide of SEQ ID NO: 2 at the C-terminal side of Lys at 313 of SEQID NO: 2.

From this experiment, it was found that there are at least two proteaseaccessible sites, Lys at 313 and Lys at 520 SEQ ID NO: 6. These twosites were mutated to other amino acid residues by saturationmutagenesis and it was found that mutations at Lys at 313 and Lys at 520of SEQ ID NO: 2 increase the insecticidal activity significantly. Forexample, the activity of the PHI-4 polypeptide variant, K313Q (MUT ID:889), was enhanced 2.3 fold over the activity of PHI-4 polypeptide ofSEQ ID NO: 2 as measured in an FAE assay (Megatable 1). The activity ofthe PHI-4 polypeptide K520Q (MUT ID: 881) was increased 3.1 fold.Activity increases were also found in combinations with other mutations.For example, the activity of the PHI-4 polypeptide having the R097D andK520E substitutions (SEQ ID NO: 52) is 3.5 fold higher than that ofPHI-4-R097D (MUT ID: 8) alone by FAE assay.

Example 16: Saturation Mutagenesis of Amino Acid Residues Selected bySite Directed Single Amino Acid Mutagenesis

Certain amino acid residues showed activity changes when mutated by sitedirected single amino acid mutagenesis. Those residues were “Selected”for saturation mutagenesis. For the purposes of this example, “Selected”can refer to single amino acid mutations that affect the activity,positively or negatively, relative to the parental backbone in whichthey were made. More specifically substitutions of Megatable 1 with MeanFAE Indices of <0.7 or of >1.3 are deemed “Selected”. For example,Selected amino acid residues were found by performing site directedmutagenesis at certain residues such as Arg and Lys. These basic aminoacid residues were mutated to either acidic (Asp, Glu) or neutral, polar(e.g.: Asn and Gin) residues, and the activity of those mutants wasdetermined by the FAE insect assay individually. Acidic amino acidresidues such as Asp and Glu were changed to basic (e.g.: Arg, Lys) orneutral, polar (e.g.: Asn and Gin) residues and the mutant activity wasdetermined. Neutral, polar amino acid residues such as Gin and Asn weremutated to either acidic (Asp, Glu) or basic (e.g.: Arg, Lys) amino acidresidues to see if the activity of those mutants were changed positively(for example mean FAE Index >1.3 relative to the reference protein) ornegatively (for example mean FAE Index <0.7 relative to the referencesequence). Another example of finding Selected amino acid residues isbased on the sequence-function relationship. Since AXMI-205 is a memberof the perforin family it is possible to identify amino acid residues ofPHI-4 polypeptide of SEQ ID NO: 2 which are involved in the mode ofaction elements such as membrane insertion initiation and receptorbinding loops. Amino acid residues found in those regions wereconsidered Selected for saturation mutagenesis in this Example. One canuse the alanine scanning to empirically define Selected residues. Thistechnique was used to find additional Selected amino acid residues inthe putative receptor binding loops.

After any amino acid residues were determined Selected, those residueswere subjected to saturation or near saturation mutagenesis to produce aset of up to 19 mutants for each site (20 all possible amino acids minusthe amino acid found in the wild type). The insecticidal activity of allthese mutants was determined by the FAE insect assay. Saturationmutagenesis of the Selected amino acid residues, was useful foridentifying substitutions with Mean FAE Indices of >1, in manycases >1.3. When the activity of one single amino acid mutation wasfound to be positive by showing increased activity over the PHI-4polypeptide of SEQ ID NO: 2, the saturation mutagenesis enabled us tofind other mutation(s) that showed further increased activity. Forexample, while the FAE Index of E082Q (MUT ID: 370) was positive (1.37),the saturation mutagenesis at this site revealed other mutations showingmuch higher FAE Indices. For example, the index of E0821 (MUT ID: 219)was 7.80 and that of E082L (MUT ID: 259) was 2.71 indicating that PHI-4polypeptide of SEQ ID NO: 2 hydrophobic residues are beneficial at thissite as far as its insecticidal activity is concerned.

Other Selected amino acid substitutions resulted in decreased activity.When these sites were examined further by saturation mutagenesis,substitutions with Mean FAE Indices of >1 were observed. For example,the FAE Indices of K099Q (MUT ID: 677), K099E (MUT ID: 715) were 0.34and 0.26, respectively. This shows that Lysine at this site isfunctionally involved in activity and that alternative substitutions mayresult in improved activity. In this example, the saturation mutagenesisrevealed substitutions with Mean FAE Index >1. For example, thesubstitution K099L (MUT ID: 299) has a Mean FAE Index of 5.72 (Megatable1). Similar instances were found across the entire PHI-4 polypeptide ofSEQ ID NO: 2, for example those indicated in Table 7. Table 7 shows theMean FAE Indices for nine pairs of substitutions. All data is fromMegatable 1.

TABLE 7 Substitution A Substitution B mean FAE mean FAE MUT IDSubstitution Index MUT ID Substitution Index 570 K074Q 0.7 215 K074E12.40 596 E203Q 0.59 288 E203T 2.18 800 R235Q 0.06 497 R235K 1.34 906K313E 0.14 889 K313Q 2.29 838 D395Q 0.03 832 D395R 1.60 784 S398A 0.11342 S398Q 1.51 629 K402Q 0.47 216 K402F 10.20 842 D403Q 0.03 251 D403Y3.03 611 D447Q 0.54 211 D447K 31.70

The serine at position 98 of SEQ ID NO: 2 was Selected by alaninescanning amino acid residues found in a region of the protein that issuspected overlap with the receptor binding loops. Point mutants withimproved potency may then be used to prepare and screen combinatoriallibraries based on that diversity.

Taking mean FAE Index <0.7 as a definition of Selected, the followingpositions are deemed Selected: P14, D24, Q38, E53, R55, R61, Q75, D76,E83, E118, E126, D152, R166, K188, K191, D193, K242, P243, R248, D254,L266, D268, A270, D274, D298, K313, D315, K316, D321, V343, S349, Q360,R361, D368, 1373, D376, F378, D379, D394, Y404, Q413, N430, Q449, D497,R500, S504. Saturation or near saturation mutagenesis at these positionscan be performed by the method of Example 7 or equivalent methods andpurified and screened the variant proteins for activity by the methodsof Example 2 & 3 or equivalent methods.

Example 17: Transgenic Expression and Activity Evaluation

The PHI-4 polypeptides of SEQ ID NOs 22-25 were cloned under control ofthe maize ubiquitin promoter (Christensen and Quail, (1996) TransgenicResearch 5:213-218) into a standard vector suitable for transformationof maize by Agrobacterium. Transgenic maize plants were produced by themethod of Example 20. Selected T0 plants were tested for susceptibilityto WCRW feeding by challenging T0 plants with WCRW larvae. After 19-21days of challenge, the roots were visually examined and root nodalinjury scores were recorded as described (Oleson J. et al J. EconomicEntomology 98:1-8; 2005). Root nodal injury scores are indicated in FIG.5. The data support the conclusion that the three PHI-4 polypeptidevariants provide measurable in planta efficacy for protection of maizetransgenic plants against WCRW. FIG. 5 shows the T0 seedlings in theV3-V4 growth stage were challenged as described (Oleson J. et al J.Economic Entomology 98:1-8; 2005) and root nodal injury scores wererecorded.

Example 18: In Planta Expression of Fusion Proteins

Localization of the protein can also play an important role in in plantaaccumulation. One can direct proteins such as PHI-4 polypeptides to thechloroplast using a chloroplast targeting peptide (CTP). Additionally,one can direct expression to the apoplastic space using fusions topeptides such as the barley alpha amylase-derived peptide (BAA; SEQ IDNO: 826). One may also direct transgenically expressed proteins forretention in the endoplasmic reticulum by fusing to both BAA and thesequence “KDEL” (SEQ ID NO: 828). Proteins can also be directed to thevacuolar space by fusion with the C terminal peptide from plantdefensins such as the maize defensin 20 C-terminal propeptide (SEQ IDNO: 824). Other functionally equivalent gene elements may be combined ina similar manner. One may also direct expression specifically to theroots with root-specific promoters. Each of these modifications may bemade separately or in combination and any given combination of elementsto improve accumulation of protein in plant tissue or in functionallyimproved efficacy of the expressed protein.

Example 19: Transformation of Maize by Particle Bombardment andRegeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aDNA molecule containing the PHI-4 polypeptide of nucleotide sequence(e.g., SEQ ID NO: 1) operably linked to an ubiquitin promoter and theselectable marker gene PAT (Wohlleben, et al., (1988) Gene 70: 25-37),which confers resistance to the herbicide Bialaphos. Alternatively, theselectable marker gene is provided on a separate DNA molecule.Transformation is performed as follows. Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% CLOROX™ bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5 cm target zone in preparation forbombardment.

Preparation of DNA

A plasmid vector comprising a nucleotide sequence (e.g., SEQ ID NO: 1)operably linked to an ubiquitin promoter is made. For example, asuitable transformation vector comprises a UBI1 promoter from Zea mays,a 5′ UTR from UBI1 and a UBI1 intron, in combination with a PinIIterminator. The vector additionally contains a PAT selectable markergene driven by a CAMV35S promoter and includes a CAMV35S terminator.Optionally, the selectable marker can reside on a separate plasmid. ADNA molecule comprising a toxin nucleotide sequence as well as a PATselectable marker is precipitated onto 1.1 m (average diameter) tungstenpellets using a CaCI₂ precipitation procedure as follows:

-   -   100 μL prepared tungsten particles in water    -   L (1 kg) DNA in Tris EDTA buffer (1 μg total DNA)    -   100 μL 2.5 M CaCl₂    -   10 μL 0.1 M spermidine

Each reagent is added sequentially to a tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 mL 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μL 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μLspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for expression of the toxin by assaysknown in the art or as described above.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts (SIGMAC-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/Lthiamine HCl, 120.0 g/L sucrose, 1.0 mg/L 2,4-D and 2.88 g/L L-proline(brought to volume with deionized H₂O following adjustment to pH 5.8with KOH); 2.0 g/L Gelrite™ (added after bringing to volume with dlH₂O); and 8.5 mg/L silver nitrate (added after sterilizing the mediumand cooling to room temperature). Selection medium (560R) comprises 4.0g/L N6 basal salts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix(1000×SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L sucrose, and 2.0 mg/L2,4-D (brought to volume with dl H₂O following adjustment to pH 5.8 withKOH); 3.0 g/L Gelrite™ (added after bringing to volume with dl H₂O); and0.85 mg/L silver nitrate and 3.0 mg/L Bialaphos (both added aftersterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/L thiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog, (1962)Physiol. Plant. 15:473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/Lsucrose, and 1.0 mL/L of 0.1 mM abscisic acid (brought to volume withpolished dl H₂O after adjusting to pH 5.6); 3.0 g/L Gelrite™ (addedafter bringing to volume with dl H₂O); and 1.0 mg/L indoleacetic acidand 3.0 mg/L Bialaphos (added after sterilizing the medium and coolingto 60 C).

Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO 11117-074),5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/Lthiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought tovolume with polished dl H₂O), 0.1 g/L myo-inositol, and 40.0 g/L sucrose(brought to volume with polished dl H₂O after adjusting pH to 5.6); and6 g/L Bacto-agar (added after bringing to volume with polished dl H₂O),sterilized and cooled to 60° C.

Example 20: Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with a toxinnucleotide sequence (e.g., SEQ ID NO: 1), the method of Zhao can be used(U.S. Pat. No. 5,981,840 and PCT Patent Publication Number WO1998/32326; the contents of which are hereby incorporated by reference).Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium under conditions wherebythe bacteria are capable of transferring the nucleotide sequence (e.g.SEQ ID NO: 1) to at least one cell of at least one of the immatureembryos (step 1: the infection step). In this step the immature embryoscan be immersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). The immature embryoscan be cultured on solid medium following the infection step. Followingthis co-cultivation period an optional “resting” step is contemplated.In this resting step, the embryos are incubated in the presence of atleast one antibiotic known to inhibit the growth of Agrobacteriumwithout the addition of a selective agent for plant transformants (step3: resting step). The immature embryos can be cultured on solid mediumwith antibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos are cultured on medium containing a selective agentand growing transformed callus is recovered (step 4: the selectionstep). The immature embryos are cultured on solid medium with aselective agent resulting in the selective growth of transformed cells.The callus is then regenerated into plants (step 5: the regenerationstep), and calli grown on selective medium can be cultured on solidmedium to regenerate the plants.

Megatable Legends

Megatable 1. The definitions of the column headings are as follows: “MUTID”, a unique identifier for each substitutions; “Backbone”, the SEQ IDcorresponding to the polypeptide backbone in which the substitution wasmade; “Position”, amino acid position according to the numberingconvention of SEQ ID NO: 2, “Ref. A.A.”, the standard single letter codefor the amino acid present in the backbone sequence at the indicatedposition; “Substitution”, the standard single letter code for the aminoacid present in the mutant sequence at the indicated position; “FAE”,the arithmetic Mean FAE Index as further defined in Example 3; “p-value”the calculated p value associated with the hypothesis that the variantpolypeptide is significantly different than the reference protein usedin that particular FAE assay, as defined further in Example 3; “EC50(ppm)”, EC50 as defined in example 3 with the EC50 dose given in ppm forthe toxin portion of the sample; “Deviation”, Mean Deviation Score asdefined in Example 4; “Example #”, the example number corresponding tothe creation of the variant. The reference protein against which thevariant protein is compared is: (MUT IDs: 1-872 and 911-1135) used SEQID NO: 6 as the reference protein; (MUT IDs: 873-910) used SEQ ID NO: 8as the reference protein.

Megatable 2. The definitions of the column headings are as follows: “SEQID NO:”, a unique identifier for each DNA or amino acid sequence;“Trivial Name”, a trivial but unique name for each DNA or proteinsequence; “Reference Protein”, the SEQ ID NO corresponding to thereference protein used in the FAE assays for each variant; “FAE”, thearithmetic Mean FAE Index as further defined in Example 3; “p-value” thecalculated p value associated with the hypothesis that the variantpolypeptide is significantly different than the reference protein usedin that particular FAE assay, as defined further in Example 3; “EC50Fold”, the fold increase in potency as defined by ratio of referenceEC50 to variant EC50 is calculated in each of multiple experiments andthen the mean of these independently measured numbers is reported as“EC50 Fold”; “Deviation”, Mean Deviation Score as defined in Example 4;“Example #”, the example number corresponding to the creation of thevariant. The reference protein against which the variant protein iscompared is: (MUT IDs: 1-872 and 911-1135) used SEQ ID NO: 6 as thereference protein; (MUT IDs: 873-910) used SEQ ID NO: 8 as the referenceprotein.

1. A variant polypeptide selected from SEQ ID NO: 148; SEQ ID NO: 225;SEQ ID NO: 226; SEQ ID NO: 227; SEQ ID NO: 228; SEQ ID NO: 229; SEQ IDNO: 73; SEQ ID NO: 230; SEQ ID NO: 149; SEQ ID NO: 231; SEQ ID NO: 232;SEQ ID NO: 233; SEQ ID NO: 150; SEQ ID NO: 74; SEQ ID NO: 234; SEQ IDNO: 235; SEQ ID NO: 236; SEQ ID NO: 151; SEQ ID NO: 237; SEQ ID NO: 183;SEQ ID NO: 75; SEQ ID NO: 238; SEQ ID NO: 76; SEQ ID NO: 77; SEQ ID NO:239; SEQ ID NO: 152; SEQ ID NO: 153; SEQ ID NO: 240; SEQ ID NO: 78; SEQID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81; SEQ ID NO: 82; SEQ ID NO: 51;SEQ ID NO: 184; SEQ ID NO: 154; SEQ ID NO: 241; SEQ ID NO: 559; SEQ IDNO: 560; SEQ ID NO: 561; SEQ ID NO: 562; SEQ ID NO: 563; SEQ ID NO: 564;SEQ ID NO: 565; SEQ ID NO: 566; SEQ ID NO: 567; SEQ ID NO: 568; SEQ IDNO: 569; SEQ ID NO: 570; SEQ ID NO: 571; SEQ ID NO: 572; SEQ ID NO: 573;SEQ ID NO: 574; SEQ ID NO: 575; SEQ ID NO: 576; SEQ ID NO: 577; SEQ IDNO: 578; SEQ ID NO: 717; SEQ ID NO: 718; SEQ ID NO: 719; SEQ ID NO: 720;SEQ ID NO: 721; SEQ ID NO: 722; SEQ ID NO: 723; SEQ ID NO: 724; SEQ IDNO: 725; SEQ ID NO: 726; SEQ ID NO: 727; SEQ ID NO: 728; SEQ ID NO: 729;SEQ ID NO: 730; SEQ ID NO: 731; SEQ ID NO: 732; SEQ ID NO: 733; SEQ IDNO: 734; SEQ ID NO: 735; SEQ ID NO: 736; SEQ ID NO: 784; SEQ ID NO: 785;SEQ ID NO: 786; SEQ ID NO: 787; SEQ ID NO: 788; SEQ ID NO: 789; SEQ IDNO: 790; SEQ ID NO: 791; SEQ ID NO: 792; SEQ ID NO: 793; SEQ ID NO: 794;SEQ ID NO: 795; SEQ ID NO: 796; SEQ ID NO: 797; SEQ ID NO: 798; SEQ IDNO: 799; SEQ ID NO: 800; SEQ ID NO: 801; SEQ ID NO: 802; and SEQ ID NO:803, wherein the variant polypeptide has at least 1.3 fold increasedinsecticidal activity against Western corn rootworm larvae compared tothe polypeptide of SEQ ID NO:
 35. 2. (canceled)
 3. (canceled) 4.(canceled)
 5. (canceled)
 6. (canceled)
 7. The PHI-4 polypeptide of claim1, wherein the insecticidal activity is increased at least 2 fold orgreater compared to the polypeptide of SEQ ID NO:
 35. 8. The PHI-4polypeptide of claim 1, wherein the insecticidal activity is increasedat least 5 fold or greater compared to the polypeptide of SEQ ID NO: 35.9. (canceled)
 10. The variant polypeptide of claim 1, wherein theimproved insecticidal activity compared to the polypeptide of SEQ ID NO:35 is quantitated as a Mean FAE Index.
 11. The variant polypeptide ofclaim 1, wherein the improved insecticidal activity compared to thepolypeptide of SEQ ID NO: 35 is quantitated as an EC50 value.
 12. Thevariant polypeptide of claim 1, wherein the improved activity comparedto the polypeptide of SEQ ID NO: 35 is quantitated as a Mean DeviationScore.
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. A polynucleotideencoding variant polypeptide of claim
 1. 17. (canceled)
 18. (canceled)19. (canceled)
 20. (canceled)
 21. (canceled)
 22. The polynucleotide ofclaim 16, wherein the insecticidal activity of the variant polypeptideis increased at least 2 fold or greater compared to the polypeptide ofSEQ ID NO:
 35. 23. The polynucleotide of claim 16, wherein theinsecticidal activity of the variant polypeptide is increased at least 5fold or greater compared to the polypeptide of SEQ ID NO:
 35. 24. Acomposition, comprising an insecticidally-effective amount of thevariant polypeptide of claim
 1. 25. A method of inhibiting growth orkilling an insect pest, comprising contacting the insect pest with thecomposition of claim
 24. 26. A method for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theresistant insect pest population with the composition of claim
 24. 27.The method of claim 26, wherein the pesticidal protein is selected fromCry1Ac, Cry1Ab, Cry1A.105, Cry1Ac, Cry1F, Cry1Fa2, Cry1F, Cry2Ab, Cry3A,mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, Cry9c, eCry3.1Ab and CBI-Bt.28. A transgenic plant or progeny thereof, comprising the polynucleotideof claim
 16. 29. The transgenic plant or progeny thereof of claim 28,wherein the transgenic plant is a monocotyledon.
 30. The transgenicplant or progeny thereof of claim 29, further comprising one or moreadditional transgenic traits.
 31. Seed, grain or processed productthereof of the transgenic plant of claim 28, wherein the seed, grain, orprocessed product thereof comprises the polynucleotide of claim
 16. 32.An expression cassette, comprising the polynucleotide of claim 16operably linked to one or more regulatory sequences directing expressionof the variant polypeptide.
 33. A transgenic plant or plant cell,comprising the expression cassette of claim
 32. 34. A method forprotecting a plant from an insect pest, comprising expressing in theplant or cell thereof, an insecticidally-effective amount of the variantpolypeptide of claim
 1. 35. A method for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of the variant polypeptide of claim 1.36. A method of inhibiting growth or killing an insect pest, comprisingcontacting the insect pest with a composition comprising aninsecticidally-effective amount of the variant polypeptide of claim 1.37. A method for controlling an insect pest population resistant to apesticidal protein, comprising contacting the insect pest populationwith an insecticidally-effective amount of the variant polypeptide ofclaim
 1. 38. A fusion protein comprising the variant polypeptide ofclaim 1.